Association between SARS-CoV-2 RNAemia, skewed T cell responses, inflammation, and severity in hospitalized COVID-19 people living with HIV

Summary Severe COVID-19 outcomes have been reported in people living with HIV (PLWH), yet the underlying pathogenetic factors are largely unknown. We therefore aimed to assess SARS-CoV-2 RNAemia and plasma cytokines in PLWH hospitalized for COVID-19 pneumonia, exploring associations with magnitude and functionality of SARS-CoV-2-specific immune responses. Eighteen unvaccinated PLWH (16/18 on cART; median CD4 T cell count 361.5/μL; HIV-RNA<50 cp/mL in 15/18) and 18 age/sex-matched people without HIV were consecutively recruited at a median time of 10 days from symptoms onset. PLWH showed greater SARS-CoV-2 RNAemia, a distinct plasma cytokine profile, and worse respiratory function (lower PaO2/FiO2nadir), all correlating with skewed T cell responses (higher perforin production by cytotoxic T cells as well as fewer and less polyfunctional SARS-CoV-2-specific T cells), despite preserved humoral immunity. In conclusion, these data suggest a link between HIV-related T cell dysfunction and poor control over SARS-CoV-2 replication/dissemination that may in turn influence COVID-19 severity in PLWH.

We hereby assessed SARS-CoV-2 RNAemia and the plasma cytokine milieu in a cohort of unvaccinated PLWH hospitalized for acute COVID-19 pneumonia as compared to HIV-negative individuals, and explored their association with the magnitude and functionality of SARS-CoV-2-specific T cell and humoral responses.

Study design and population
In this cross-sectional study, 18 SARS-CoV-2-unvaccinated PLWH and 18 age-and sex-matched HIV-negative individuals hospitalized for acute COVID-19 pneumonia were consecutively enrolled between March 2020 and January 2021 at one of the participating Infectious Diseases centers in Northern Italy, at a median time of 10 (IQR: 6.75-13) days after symptoms onset.
Participants characteristics are reported in Table 1.The two study groups were comparable in terms of age [60 (IQR: 48-67) years], sex, ethnicity, and comorbidities.PLWH had a lower arterial partial pressure of oxygen (P a O 2 )/fraction of inspired oxygen (F i O 2 ) nadir (p = 0.0005) compared to HIV-negative individuals.One in-hospital death (5.6%) was registered among PLWH compared to no one in the HIVnegative control group, yet the difference was not statistically significant.
Taken together, these data suggest that PLWH with COVID-19 pneumonia, especially those with an imbalanced immune system and impaired respiratory function, have a limited control of SARS-CoV-2 replication/dissemination.

Distinct plasma cytokine profile in PLWH
Next, we aimed to investigate whether PLWH also had an exacerbated cytokine storm compared to the HIV-negative individuals by measuring a panel of 12 plasma cytokines with a commercially available cytometric bead array (CBA).
To this end, we firstly performed a principal component analysis (PCA) in order to examine the influence of all the cytokines simultaneously in detecting patterns of cytokine variation between the two study groups.We thereby found a clear separation between PLWH and HIV-negative individuals based on the principal component (PC) scores (Figure 2A).The loading values assessment displayed distinct cytokines clusters (Figure 2B), suggesting that some of them had a prominent effect in determining between-group differences.
Altogether, these data indicate that, compared to HIV-negative individuals, PLWH with acute COVID-19 pneumonia have a distinct plasma cytokine pattern with higher levels of GM-CSF, TNF-a, IL-4, IL-5, and IL-17A, yet low circulating IL-2 and IL-9.This peculiar cytokine profile is associated with a poor control of SARS-CoV-2 replication/dissemination and a worse clinical severity in PLWH.

Comparable T cell activation yet higher perforin production by pro-cytolytic T cells in PLWH
Having shown in PLWH a greater SARS-CoV-2 RNAemia with a distinct plasma cytokine profile, we next aimed to assess T cell activation and pro-cytolytic phenotypes via flow cytometric analysis of peripheral blood mononuclear cells (PBMCs).CD4 and CD8 T cell activation (as defined by coexpression of CD38 and HLA-DR) was comparable in PLWH and HIV-negative individuals (Figure 4A).Likewise, percentages of pro-cytolytic CD4 and CD8 T cells (defined as either GRZB+, PRF+, or GRZB+PRF+) were similar in the two groups (Figure 4B).However, when assessing intracellular perforin (PRF) and granzyme-B (GRZB) expression (median fluorescence intensity, MFI) by these cells, we found higher PRF production by both CD4 and CD8 pro-cytolytic T cells in PLWH (PRF+ T cells: p = 0.0229, p = 0.0005; GRZB+FRF+ T cells: p = 0.0027, p = 0.0001) (Figure 4C), despite similar GRZB expression (Figure 4D).
Interestingly, PRF production by pro-cytolytic T cells appeared to be positively correlated with SARS-CoV-2 RNAemia as well as plasma GM-CSF and IL-4, yet negatively with plasma IL-2 and IL-9 as well as P a O 2 /F i O 2 nadir (Figure 3; Table S1).
These data indicate that PLWH hospitalized for SARS-CoV-2 infection do not display higher T cell activation compared to HIV-negative controls, albeit showing increased PRF production by circulating total pro-cytolytic T cells.Such elevated expression of a cytotoxic program in circulating T cells is associated with higher SARS-CoV-2 replication/dissemination, a distinct plasma cytokine milieu and worse respiratory function in PLWH.

Lower magnitude and polyfunctionality of SARS-CoV-2-specific T cell responses in PLWH
To further profile the immune status of PLWH, and considered that T cell dysfunction is a hallmark of HIV infection, we next sought to assess SARS-CoV-2-specific T cells in order to determine whether the T cell compartment of the immune system adequately responded to the infection.To this end, we evaluated both quantitatively and qualitatively SARS-CoV-2-specific T cell responses by means of a flow cytometric intracellular cytokine staining (ICS) assay upon stimulation of PBMCs with a pool of 15-mer peptides (S, N, and M) of the wild-type virus.
Altogether, these data point to a skewed T cell response in PLWH during acute COVID-19 pneumonia, with lower magnitude and polyfunctionality of SARS-CoV-2-specific T cells.Such impaired virus-specific T cell response is linked to higher SARS-CoV-2 RNAemia, systemic inflammation, and disease severity.

Preserved SARS-CoV-2-specific humoral responses in PLWH
Having found blunted SARS-CoV-2-specific T cell responses in PLWH, we next assessed humoral immunity by measuring anti-RBD total antibodies, by means of ELISA, 54,68 and Spike-ACE2 binding inhibition activity, via a receptor-binding inhibition assay. 17,54,68,69oth anti-RBD antibodies and Spike-ACE2 binding inhibition activity were comparable in the two groups (Figures 6A and 6B), positively correlating with each other (r = 0.4021, p = 0.0334) (Figure 6C).SARS-CoV-2-specific humoral responses did not appear to correlate with CD4 T cell counts in PLWH (data not shown).Moreover, no correlations were observed between humoral responses and SARS-CoV-2 RNAemia, plasma cytokines and P a O 2 /F i O 2 nadir (Figure 3; Table S1).
These finding indicate that, despite a skewed T cell immunity, PLWH display SARS-CoV-2-specific humoral responses comparable to HIV-negative people during acute COVID-19 pneumonia.

DISCUSSION
In this cross-sectional study we sought to investigate SARS-CoV-2 RNAemia, plasma cytokine milieu, T cell activation and pro-cytolytic phenotypes, as well as SARS-CoV-2-specific T cell and humoral responses in unvaccinated PLWH hospitalized for acute COVID-19 pneumonia compared to people without HIV.[5][6][7][8][9][10][11][12][13]70,71 We found that, compared to HIV-negative individuals, PLWH feature higher SARS-CoV-2 RNAemia, which is negatively correlated with CD4 T cell percentages yet positively with CD8 T cell frequencies.Since SARS-CoV-2 RNAemia has been previously shown to reflect a higher viral replication in the respiratory tract 51 as well as dissemination of SARS-CoV-2 virions in peripheral blood, 52 our finding suggest that PLWH may experience poor control over SARS-CoV-2 replication/dissemination, which is seemingly associated to the HIV-driven immune imbalances. 72,73Accordingly, as previously described in patients with immune suppression due to other causes, [74][75][76] a delayed SARS-CoV-2 clearance in the upper respiratory tract has been proven in PLWH with severe T cell depletion. 64,67Likewise, in a murine model of acute SARS-CoV-2 infection, CD4 T cells depletion led to delayed viral clearance, 77 highlighting the pivotal role of CD4 T cells in controlling viral replication.In addition, we showed that SARS-CoV-2 RNAemia appears to be negatively correlated with P a O 2 /F i O 2 nadir, which is significantly lower in PLWH, pointing to a link between viral RNAemia and worse respiratory function.Such observation agrees with previous findings in the general population, as SARS-CoV-2 RNAemia has been clearly associated with severe disease and worse clinical outcomes, 52,57,58,63 yet expands our knowledge to PLWH.
Data regarding COVID-19-related cytokine storm in PLWH are limited and controversial, variously reporting lower 33,50 or higher 8,78 levels of inflammatory markers.We hereby displayed that PLWH with acute COVID-19 feature a distinct plasma cytokine profile (with higher Th1/ Th2/Th17-like cytokines, yet lower IL-2 and IL-9), which, in turn, is associated with poorer control of SARS-CoV-2 replication/dissemination and worse respiratory insufficiency.These data are in accordance with previous studies in HIV-negative individuals, which reported higher peripheral inflammation to be associated with both SARS-CoV-2 RNAemia 14,54,57 and clinical severity. 14,41,44mmunoprofiling of total circulating T cells revealed higher intracellular PRF production by cytotoxic T cells yet similar T cell activation in PLWH compared to HIV-negative individuals.Whereas several studies have shown a hyperactivated T cell phenotype in PLWH compared to healthy controls, [79][80][81] as well as in individuals with severe COVID-19 compared to those with mild disease, [82][83][84][85][86][87] few data exist on the impact of HIV/SARS-CoV-2 coinfection on T cell activation, with one case report suggesting that SARS-CoV-2 infection may not further increase T cell activation in PLWH, 88 while another study reporting higher CD38+HLA-DR+ CD4 and CD8 T cells in PLWH with SARS-CoV-2 infection. 26herefore, the underlying mechanisms by which in our cohort PLWH with COVID-19 pneumonia showed similar T cell activation compared to HIV-negative controls remain to be elucidated.
We also showed that, while HIV-negative individuals develop a multi-layered SARS-CoV-2-specific immune response involving both T cell and humoral compartments at 10 days after the symptoms onset-which correspond to the temporal bridging between innate and adaptive immunity-, PLWH mount what seems to be a less coordinated adaptive immune response with Spike-binding and Spike-blocking antibodies comparable to HIV-negative controls, but lower magnitude and polyfunctionality of SARS-CoV-2-specific T cells.
Prior studies assessing adaptive immunity to SARS-CoV-2 in PLWH yielded apparently conflicting findings, which yet fit with our observations when critically considered as a whole.Two previous reports described no differences according to HIV status in SARS-CoV-2-specific T cell responses in individuals recovered from SARS-CoV-2 infection, 26,27 albeit a positive correlation of SARS-CoV-2-specific T cell responses with CD4 T cell counts 36 and CD4/CD8 ratio 26 was found in PLWH.Our cohort of PLWH features lower median CD4 T cell counts and CD4/ CD8 ratio than those of the aforementioned, likely explaining the skewed T cell responses to SARS-CoV-2 infection.Another study found that while virally suppressed PLWH develop similar SARS-CoV-2-specific T cell responses compared to people without HIV, those with detectable HIV viremia have significantly lower frequencies of polyfunctional antigen-specific T cells as well as impaired ability to cross-recognize viral variants. 35However, our cohort included only three virally unsuppressed PLWH, thus not allowing to specifically assess the contribution of uncontrolled HIV replication in hampering T cell responses against SARS-CoV-2.
32]37 However, in agreement with our data, other three recent studies proved that T cell and humoral immunity to SARS-CoV-2 do not necessarily move in the same direction, by showing that, when compared to HIVnegative individuals, convalescent PLWH developed similar humoral responses, but lower SARS-CoV-2-specific T cells. 33,34,39Nevertheless, the majority of aforementioned studies included individuals in the convalescent period following predominately mild disease, while our study focused on PLWH hospitalized during the acute phase of severe COVID-19, thus providing insights in such specific clinical setting.
What is more, we found that blunted SARS-CoV-2-specific T cell responses-which characterize PLWH-are associated with higher RNAemia, exacerbated cytokine storm, as well as lower P a O 2 /F i O 2 nadir, while no significant correlations were found between these features and humoral responses.These data align with current evidence: while protection from infection is mediated primarily by neutralizing antibodies, protection from severe COVID-19 is mediated by a coordinated presence of antibody and T cellular immunity. 16,17,19,89,90 mounted robust antibody responses but had undetectable circulating SARS-CoV-2-specific T cells; on the contrary, those with a rapid expansion of IFN-g+ SARS-CoV-2-specific T cells rapidly controlled infection without developing severe disease. 172][93] In light of these evidences, our data suggest that impaired SARS-CoV-2 specific T cell responses in PLWH may contribute to a delayed viral clearance with possible systemic dissemination, further fueling peripheral inflammation.However, even though prior research demonstrated that a delayed engagement of antiviral immune defenses can contribute to SARS-CoV-2 dissemination via the bloodstream to distal organs, 53 the cross-sectional nature of our study does not allow to establish causality.Therefore, we cannot rule out an alternative pathogenetic model in which a higher ab initio viral burden, characterized by increased SARS-CoV-2 RNAemia, might hinder the development of an effective SARS-CoV-2-specific T cell response.It is worth noting that these two models are not mutually exclusive and could potentially reinforce each other, creating a self-perpetuating cycle.Additionally, differing from T cell activation, PRF production by pro-cytolytic T cells-which is higher in PLWH-was associated with higher SARS-CoV-2 RNAemia, higher plasma cytokines and lower P a O 2 /F i O 2 nadir.These observations fit with previous literature, which reported elevated expression of a cytotoxic program in circulating T cells in SARS-CoV-2-infected patients with higher systemic inflammation 94 and severe disease. 85,86,92,95,96Although we have not identified the antigenic specificity of such T cells, increases in T cells with a cytotoxic potential may result in an altered T cell landscape that could affect tissue integrity, depending on their trafficking capabilities.
Interestingly, in sharp contrast to other cytokines, we found that plasma IL-2 and IL-9 are significantly lower in PLWH.This cytokine pattern also associates with lower SARS-CoV-2-specific T cells and higher PRF production by cytotoxic T cells, as well as greater SARS-CoV-2 RNAemia and lower P a O 2 /F i O 2 nadir.8][99] Furthermore, previous studies reported higher circulating IL-2 levels as a fingerprint of asymptomatic/mild COVID-19. 100,1016][107] By contrast, the roles of IL-9 in HIV and SARS-CoV-2 infections have not been specifically characterized to date, thus the underlying reasons for our findings concerning plasma IL-9 are unclear and remain to be elucidated.It should also be noted that, while intracellular production of TNF-a, IL-4, and IL-17A by SARS-CoV-2-specific T cells was lower in PLWH, the plasma cytokine analysis revealed an opposite trend for the same cytokines, with lower levels in HIV-negative individuals.While such findings may appear contradictory, it must be considered that the former represent the specific antiviral response, which exert a protective effect, while the latter are expression of the cytokine storm sustained by both T lymphocytes and non-specific immune cells, which is associated with impaired viral clearance and worse respiratory insufficiency.In conclusion, our data show that PLWH with COVID-19 pneumonia, despite preserved humoral immunity, mount skewed T cell responses with lower magnitude and polyfunctionality of SARS-CoV-2-specific T cells.This faulty virus-specific T cell response is closely intertwined with higher SARS-CoV-2 RNAemia, systemic inflammation, and disease severity.These findings suggest that additional measures to reduce the viral burden during early SARS-CoV-2 infection may be warranted in this high-risk population, and support prioritization of PLWH for SARS-CoV-2 vaccination in order to enhance T cell immunity and therefore prevent severe disease.

Limitations of the study
Some limitations need to be acknowledged in this study.Firstly, despite clear dissimilarities in viral and immunological aspects, we did not find any statistically significant difference in mortality between the two groups, likely owing to the lack of power due to the small sample size.Secondly, study participants were enrolled during the first two waves of the COVID-19 pandemic, when the currently circulating viral variants had not yet emerged and vaccines were not available, so immune responses to the Omicron variant, which is now the most widespread all over the world, as well as the role of the vaccination and hybrid immunity, cannot be inferred.Furthermore, since the study population is primarily an older one, the findings of this study are not necessarily generalizable to a younger population.Additionally, due to the exploratory nature of this study and its small sample size, no adjustment for multiple comparison was made, so our findings need to be confirmed in larger studies.Lastly, as per cross-sectional nature of this study, all measurements were only available at a single time point during hospitalization, so that no kinetics can be derived, limiting the possibility of characterizing the dynamics of the development of immune responses in relation to SARS-CoV-2 RNAemia and systemic inflammation and, thus, establishing causality.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
This study involved human participants, whose characteristics are described in Tables 1 and 2.
The study was approved by the local Ethics Committee (Comitato Etico ASST Santi Paolo e Carlo: 2020/ST/049, 2020/ST/049_BIS); written informed consent was obtained from each participant.All research was performed in accordance with the Declaration of Helsinki.

Study design and population
In this cross-sectional study we consecutively enrolled unvaccinated PLWH hospitalized for ascertained SARS-CoV-2 infection (positive RT-PCR nasopharyngeal swab) and radiologically documented pneumonia at one of the participating Infectious Diseases centers in Northern Italy.Age-and sex-matched HIV-negative individuals with COVID-19 pneumonia requiring hospitalization were also enrolled as controls.Demographic and clinical characteristics of the study participants as well as HIV-related features of PLWH were collected.P a O 2 /F i O 2 nadir, i.e. the ratio between arterial partial pressure of oxygen (P a O 2 ) and fraction of inspired oxygen (F i O 2 ) at its lowest point throughout the hospitalization, was used as a marker of the degree of respiratory insufficiency.

PBMCs and plasma isolation
Peripheral blood samples were collected in EDTA tubes from all study participants.Plasma was separated by centrifugation and stored at -

Immunophenotyping
T cell activation and pro-cytolytic phenotype were determined by flow cytometry.Briefly, 1.5310 6 thawed PBMCs were plated in complete RPMI containing 10% human serum supplemented with 1% penicillin-streptomycin-glutamine.Overnight-rested PBMCs were stained with the appropriate surface antibodies for 20 minutes at 4 C in the dark; after 2% paraformaldehyde (PFA) fixation for 30 minutes at 4 C, cells were permeabilized with 0.2% saponin (Sigma-Aldrich) and stained for intracellular markers (Granzyme-B and Perforin) for 30 minutes at room temperature.Cells were then washed and resuspended in 500 mL of phosphate buffered saline (PBS).Dead cells were labeled using Viobility Fixable Dye (Miltenyi Biotec).Antibodies used were: CD4-APC-Vio770, CD8-APC, HLA-DR-VioBlue, CD38-PE-Vio770, Granzyme-B-PE and Perforin-FITC (Miltenyi Biotec).Samples were acquired using FACSVerseä cytometer (BD Biosciences) and FCS files were analyzed using FlowJo 10.8 (BD Biosciences).Activated T cells were defined as CD38+HLA-DR+, whereas pro-cytolytic (cytotoxic) T cells -i.e., T cells with pre-formed intracellular cytotoxic molecules -as either Granzyme-B(GRZB)+, Perforin(PRF)+, or GRZB+PRF+.GRZB and PRF were gated separately and then their coexpression was determined by using the FlowJo Boolean Gating tool (combination gates).Intracellular GRZB and PRF production by pro-cytolytic T cells was quantified by means of median fluorescence intensity (MFI).Representative plots are shown in supplemental information (Figure S1).

Intracellular cytokine staining (ICS) assay
SARS-CoV-2-specific T cell responses were measured by means of a flow cytometric intracellular cytokine staining (ICS) assay.Briefly, 1.5310 6 thawed PBMCs were plated in complete RPMI containing 10% human serum supplemented with 1% penicillin-streptomycin-glutamine.Overnight-rested PBMCs were stimulated for 5 hours with a pool of commercially available 15-mer peptides (1 mg/mL) covering the immunodominant sequence domain of the wild-type Spike (S) protein, the complete sequence of the Nucleocapsid (N) protein, and the complete sequence of the Membrane (M) protein (PepTivator SARS-CoV-2, Miltenyi Biotec).Phorbol myristate acetate (PMA) and ionomycin (Sigma-Aldrich) were used as positive control (25 ng/mL and 1 mg/mL, respectively), whereas negative controls were left untreated.Brefeldin (1 mg/mL) was added after 1 h of stimulation.Cells were harvested and stained for surface markers 20 minutes at 4 C in the dark; after 2% PFA fixation, cells were permeabilized with 0.2% saponin (Sigma-Aldrich) and stained for intracellular cytokines for 30 minutes at room temperature.Dead cells were labeled using Viobility Fixable Dye (Miltenyi Biotec).Antibodies used were: CD4-APC-Vio770, CD8-PerCP-Vio700, IL-17A-FITC, IL-4-PE, TNF-a-PE-Vio770, IFN-g-VioBlue, IL-2-APC (Miltenyi Biotec).Samples were acquired using FACSVerseä cytometer (BD Biosciences) and FCS files were analyzed using FlowJo 10.8 (BD Biosciences).SARS-CoV-2-specific T cells were determined subtracting unspecific background activation in paired unstimulated samples (negative control) from stimulated samples; negative values were set to zero.SARS-CoV-2-specific T cells were expressed by percentage and integrated median fluorescence intensity (iMFI), a metric which incorporates both magnitude (frequency of cytokine-producing cells) and quality (MFI, which quantify cytokine production by such cells) of an immune response, thus reflecting the total functional response of a population of cytokine-producing T cells.T cell polyfunctionality was assessed by using the FlowJo Boolean Gating tool (combination gates) and SPICE 6.0 to identify single-, dual-, triple-cytokine-producing SARS-CoV-2-specific Th1 cells.Representative plots are shown in supplemental information (Figure S2).

Anti-RBD antibodies
Total anti-RBD antibodies were determined in plasma samples by a homemade ELISA.Briefly, high-binding 96-well plates (Greiner Bio-One) were coated with 3 mg/mL of recombinant wild-type SARS-CoV-2 receptor binding domain (RBD) (ACROBiosystems) diluted in 0.5 mM of carbonate-bicarbonate buffer pH 9.6 (Sigma-Aldrich) and incubated overnight at 4 C. Plates were washed with PBS-0.05%Tween-20 and blocked for 1 hour with PBS-2%BSA at 37 C. Plasma samples were serially diluted in PBS-1%BSA in triplicates (1:40, 1:240 and 1:1440), added to plates, and incubated for 2 hours at 37 C. A mix of biotinylated goat anti-human k and l light chain were used at 1:2500 (Bethyl Laboratories, Inc., A80-115B and A80-116B) for detection, followed by avidin-HRP diluted at 1:2000 (ThermoFisher), for 30 minutes at room temperature in the dark and mild agitation.The detection was carried out with 3,3',5,5'-tetramethylbenzidine (TMB) (Invitrogen) and quenched with 1 M H 2 SO 4 .Two plasma samples collected before the SARS-CoV-2 pandemic were included as negative controls, whereas an RBD-specific monoclonal antibody (Human Anti-SARS-CoV-2 Spike RBD Monoclonal Antibody, clone BIB116, Creative Diagnostics) was included as positive control.The optical density (OD) was measured by using TECAN Sunriseä at 450 nm and 620 nm, and the area under the curve (AUC) was determined with GraphPad Prism 9.4.

Spike-ACE2 binding inhibition activity
A Spike-ACE2 inhibition assay was used to measure the ability of antibodies to block the interaction between the spike protein RBD and the ACE2 receptor, thus estimating potential viral neutralization activity, as previously described.Briefly, high-binding 96-well plates (Corning) were coated with 2 mg/mL of recombinant human ACE2-Fc (InvivoGen) diluted in 100 mM carbonate-bicarbonate buffer pH 9.6 (Sigma-Aldrich) and incubated overnight at 4 C. Plates were washed with PBS-0.05%Tween-20 and blocked with PBS-2%BSA for 1 hour at room temperature.Plasma samples were diluted 1:20 in triplicates in PBS-1%BSA and incubated with 12 ng of recombinant wild-type SARS-CoV-2 RBD-HRP (ACROBiosystems) for 1 hour at 37 C. Plates were washed and incubated with the pre-incubated plasma and RBD-HRP for 1 hour at room temperature, then detected with TMB and 1 M H 2 SO 4 .RBD-HRP alone (negative control) and plasma samples of the pre-COVID-19 era with no RBD-HRP (positive controls) were also included.The OD was measured by using EnSight (Multimode Plate Reader, PerkinElmer) at 450 nm and 570 nm.The results were expressed as percentage (%) of inhibition, calculated as [(1 -sample OD)/average negative control OD)]3100.

QUANTIFICATION AND STATISTICAL ANALYSIS
Continuous variables were expressed as median (interquartile range, IQR), while categorical variables as number, n (percentage, %).Mann-Whitney U test was used for comparisons between groups for continuous variables.Fisher exact test was employed for comparison of categorical variables.Spearman's correlation test was used for correlations between continuous variables.Principal Component Analysis (PCA) was used to visualize plasma cytokines clustering patterns in the two study groups.Data were analyzed and graphed with GraphPad Prism 9.4.Permutation test in SPICE 6.0 was employed to compare polyfunctionality patterns of SARS-CoV-2-specific Th1 cells in the two groups.P values less than 0.05 were considered statistically significant.

Table 1 .
Demographic and clinical characteristics of study participants

Table 2 .
HIV-related characteristic of PLWH MSM, men who have sex with men; MSW, men who have sex with women; WSM, women who have sex with men; IDU, injective drugs use; cART, combination antiretroviral therapy; INSTI, integrase strand transfer inhibitor; PI, protease inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor.
Kinetic studies assessing immune responses throughout SARS-CoV-2 infection demonstrated that patients with prolonged infection and severe disease Figure 1.SARS-CoV-2 RNAemia and correlations with immunological parameters (A) Plasma SARS-CoV-2 RNAemia in PLWH and HIV-negative individuals.Red/blue dots: individual values; bar: median; error bars: interquartile range; statistical analysis: Mann-Whitney U test (p values < 0.05 reported above the line connecting the two groups).(B)Correlation between SARS-CoV-2 RNAemia and percentage of CD4 T cells (within lymphocytes).(C)Correlation between SARS-CoV-2 RNAemia and percentage of CD8 T cells (within lymphocytes).Red circle: PLWH with HIV-RNA <50 copies/mL and CD4 T cell count R200/mL; open red circle: PLWH with HIV-RNA R50 copies/mL and/or CD4 T cell count <200/mL; blue circle: HIV-negative controls; dashed line: simple linear regression line; statistical analysis: Spearman's correlation test (r and p value reported above each plot).PLWH: n = 18; HIV-negative individuals: n = 18.
80 C. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation (Lymphosep medium, Biowest), cryopreserved in fetal bovine serum (EuroClone) with 10% Dimethyl Sulfoxide (EuroClone), and stored at -80 C and then in liquid nitrogen.Quantitative assessment of SARS-CoV-2 RNA was performed on frozen plasma.Briefly, viral RNA was extracted from 140 mL of plasma by using the QIAamp Viral RNA Mini Kit (QIAGEN), following the manufacturer's instructions.5 mL of extracted RNA was quantified by realtime PCR using the CDC 2019-nCoV_N1 primers and probe set (Centers for Disease Control and Prevention, CDC, update June 2020) and the TaqPathä 1-Step RT-qPCR Master Mix CG (ThermoFisher).For absolute quantification, 10-fold dilutions of the 2019-nCoV_N Positive Control plasmid (Integrated DNA Technologies) were used to generate a standard curve.The assay was run in duplicate for each sample and a non-template control well was included as negative control.Quantification of the RPP30 gene was performed to determine the quality of RNA extraction.