An Early Th1 Response Is a Key Factor for a Favorable COVID-19 Evolution

The Th1/Th2 balance plays a crucial role in the progression of different pathologies and is a determining factor in the evolution of infectious diseases. This work has aimed to evaluate the early, or on diagnosis, T-cell compartment response, T-helper subsets and anti-SARS-CoV-2 antibody specificity in COVID-19 patients and to classify them according to evolution based on infection severity. A unicenter, randomized group of 146 COVID-19 patients was divided into four groups in accordance with the most critical events during the course of disease. The immunophenotype and T-helper subsets were analyzed by flow cytometry. Asymptomatic SARS-CoV-2 infected individuals showed a potent and robust Th1 immunity, with a lower Th17 and less activated T-cells at the time of sample acquisition compared not only with symptomatic patients, but also with healthy controls. Conversely, severe COVID-19 patients presented with Th17-skewed immunity, fewer Th1 responses and more activated T-cells. The multivariate analysis of the immunological and inflammatory parameters, together with the comorbidities, showed that the Th1 response was an independent protective factor for the prevention of hospitalization (OR 0.17, 95% CI 0.03–0.81), with an AUC of 0.844. Likewise, the Th1 response was found to be an independent protective factor for severe forms of the disease (OR 0.09, 95% CI: 0.01–0.63, p = 0.015, AUC: 0.873). In conclusion, a predominant Th1 immune response in the acute phase of the SARS-CoV-2 infection could be used as a tool to identify patients who might have a good disease evolution.


Introduction
The SARS-CoV-2 infection is characterized by a wide spectrum of clinical profiles: an asymptomatic process, mild coronavirus disease (COVID-19) (with or without need for hospitalization) and severe COVID-19 with complications that can lead to the death of the patient [1]. Several characteristics such as male gender, genetic susceptibility, age,

Study Definitions
COVID-19 case was defined as a positive result for SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) assay performed on a nasal swab sample from patients in whom COVID-19 was suspected.

Study Definitions
COVID-19 case was defined as a positive result for SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) assay performed on a nasal swab sample from patients in whom COVID-19 was suspected.
Ventilatory failure was defined as a SaO 2 /FiO 2 < 300 (blood oxygen pressure/fractional inspired oxygen), or the need for mechanical ventilation (either non-invasive positive pressure ventilation or invasive mechanical ventilation). Poor outcome was defined as the presence of at least one of the following criteria: (a) ventilatory failure; (b) admission to the intensive care unit (ICU); or (c) death during admission by any cause.
Lymphopenia was defined as a total lymphocyte count of less than 0.85 cells/mL. Comorbidities are medical conditions associated with a higher risk of becoming severely ill from COVID-19 [40]. Patients with comorbidities were considered to be those with a history of diabetes mellitus, obesity, arterial hypertension, dyslipidemia, acute myocardial infarction, advanced chronic kidney disease, active smoking or ex-smokers. Association of the presence of comorbidities with the evolution was studied jointly (any comorbidity) and also individually for each condition.
Advanced chronic kidney disease is defined as a glomerular filtration rate less than 30 mL/min. It includes stages 4 and 5 of Chronic kidney disease as defined in the K/DOQI clinical practice guidelines for chronic kidney disease [41].

Data Collection
An anonymized database was created. It contained the patient data, including demographic, clinical and laboratory data from the electronic medical record. Laboratory parameters included D-dimer (DD), lactate dehydrogenase (LDH), C reactive protein (CRP) and the number of lymphocytes.

Samples
Plasma and EDTA-treated blood samples were collected in the first 24h after the admission in the Emergency Department with a median of 7 days from the onset of the symptoms in the case of the symptomatic COVID-19 patients.

Statistical Analysis
The results in the discrete variables were shown as a frequency and a percentage. Comparisons were made with Pearson's Chi-square test (or Fisher's exact test). Odds ratio represents the relative measure of an effect.
Results of the continuous variables were expressed as median accompanied by the interquartile range in brackets. Mann-Whitney U test and Kruskal-Wallis test were used for comparisons.
Multivariate analyses were performed through a logistic regression model using variables that had presented a p-value < 0.12 in a previous univariate analysis.
Center-scaled variables were used for hierarchical clustering analysis and heatmap representation using ComplexHeatmap R package.
Probabilities under 0.05 were considered significant. Data were analyzed with Med-Calc for Windows version 19.3 (MedCalc Software, Ostend, Belgium).

Population Characteristics and Biochemical Markers
Median age of the COVID-19 patients was 56 (43-69.5) years without significant differences in the proportion of men and women (56.5% men). No significant differences were found between COVID-19 patients and healthy controls in relation to age and gender (data not shown).
Once COVID-19 patients were divided according to hospitalization requirements, we observed that non-hospitalized patients were significantly younger than hospitalized patients: median 48.5 years vs. 58.5 (p = 0.004). Gender distribution according to hospitalization requirements showed that males presented higher rates of hospitalization compared to females (63% men, p = 0.038) (Table 1). However, it should be mentioned that the biggest proportion of men was found in patients with severe disease (Figure 2), especially in deceased patients (75% men, p = 0.021). The study of the inflammatory parameters (Table 1) showed the differences between non-hospitalized and hospitalized COVID-19 patients. Patients who fulfilled hospitalization requirements had higher median levels of LDH: 336 U/L (225.5-402.2) vs. 276.5 (222.5-310) (p = 0.001), CRP: 6 mg/dL (3.7-11) vs. 2.9 (1.3-5.2) (p < 0.001). DD levels were higher in hospitalized patients, but these differences were not significant compared to non-hospitalized patients: DD: 691 ng/mL (410-1414) vs. 555 (299.5-907.5) (p = 0.058). However, the comparison of DD was biased, as the number of patients in whom DD levels were evaluated as low as DDs were only tested in those patients with a poor clinical profile prognosis.
No cases of this complication were observed in non-hospitalized patients when ARDS rates were studied. The study of the inflammatory parameters (Table 1) showed the differences between non-hospitalized and hospitalized COVID-19 patients. Patients who fulfilled hospitalization requirements had higher median levels of LDH: 336 U/L (225.5-402.2) vs. 276.5 (222.5-310) (p = 0.001), CRP: 6 mg/dL (3.7-11) vs. 2.9 (1.3-5.2) (p < 0.001). DD levels were higher in hospitalized patients, but these differences were not significant compared to non-hospitalized patients: DD: 691 ng/mL (410-1414) vs. 555 (299.5-907.5) (p = 0.058). However, the comparison of DD was biased, as the number of patients in whom DD levels were evaluated as low as DDs were only tested in those patients with a poor clinical profile prognosis.
No cases of this complication were observed in non-hospitalized patients when ARDS rates were studied.

Specificity of Anti-SARS-CoV-2 Antibodies
A total of 75 (51.7%) patients developed a positive antibody response with the presence of at least one type of the tested IgG antibody against SARS-CoV-2 virus in the early stage of the disease. Non-hospitalized patients presented similar positive rates compared to hospitalized patients (51% vs. 45.6%; p = 0.076). Hence, the association between hospitalization requirements and humoral response did not show any statistical significance (Table S1).
Similarly, the production of anti-SARS-CoV-2 antibodies in asymptomatic patients was evaluated against symptomatic and severe patients, respectively. The rates of production of antibodies were similar in both comparisons (data not shown).  Table 2).

Specificity of Anti-SARS-CoV-2 Antibodies
A total of 75 (51.7%) patients developed a positive antibody response with the presence of at least one type of the tested IgG antibody against SARS-CoV-2 virus in the early stage of the disease. Non-hospitalized patients presented similar positive rates compared to hospitalized patients (51% vs. 45.6%; p = 0.076). Hence, the association between hospitalization requirements and humoral response did not show any statistical significance (Table S1).
The comparison of the Th response between healthy controls and non-hospitalized and hospitalized patients, respectively, is shown in Figure 5.
No significant results were observed when comparing the Th polarization between asymptomatic and healthy controls, hospitalization requirements and non-hospitalized patients (see Supplementary Table S3 and Figures S5 and S6, respectively).

Th1, Th2 and Th17 Subsets in Asymptomatic COVID-19 Patients Compared to Different Clinical Profiles
When the differences in Th responses were analyzed according to the progression of the disease at the acute phase of the infection (Figure 1), a higher Th1 response (quantified as total percentage of Th1 cells) of asymptomatic patients was observed compared to the severe ones: the median proportion of Th1 cells was 7.5% (5.3-10.2) vs. 4.1% (2.7-5.9) (p = 0.015, Figure 7F).

Analysis of Comorbidities in COVID-19 Patients
The association of comorbidities and hospital admission is represented in Table 3. No significant association was observed between the presence of comorbidities and the requirement for hospital admission. However, in hospitalized patients, the presence of comorbidities was associated with a higher risk of requiring ICU treatment (OR: 3.29, 95% CI: 1.09-9.95, p = 0.031). After the evaluation of each comorbidity separately, only obesity behaved as a significant risk factor for admission to the ICU (OR: 3.44; 95% CI: 1.10-10.83; p = 0.029).

Multivariate Analysis in COVID-19 Disease
Several different multivariate analyses that included the significant immunological and inflammatory variables obtained in a previous univariate analysis (Table 4) were performed in order to determine the relevance of the type of immune response in COVID-19 severity in relation to other significant variables associated with a poor evolution. The proportion of Th1 (OR: 0.22, 95% CI: 0.06-0.77, p = 0.018) in the early immune response to SARS-CoV-2 infection and higher levels of LDH (OR: 1.1, 95% CI: 1-1.1, p = 0.016) were identified as a protection and risk factor, respectively, for hospitalization requirements with an area under the curve ROC of 0.802 (95% CI AUC: 0.723-0.867, Table 4A).     Table 4B).
A third multivariate analysis was performed to study the differences between nonhospitalized patients and those patients who required hospitalization with mild to moderate symptoms (Table 4C). The activation status of CD4+ T cells (OR: 2.58, 95% CI: 1.12-2.95, p = 0.026) was a significant and independent factor with an area under the curve ROC of 0.762 (95% CI AUC: 0.334-0.841).
A fifth analysis was performed to compare mild to moderate patients with their severe counterparts (Table 4E). The presence of at least one comorbidity (OR: 5.083, 95% CI: 1.54-16.75, p = 0.007) and high levels of LDH (OR: 1, 95% CI: 1-1.01, p = 0.013) were significant and independent risk factors for a poor prognosis. Nonetheless, the proportion of senescent CD4+ T cells (OR: 0.11, 95% CI: 0.02-0.64, p = 0.014) and the proportion of quiescent Th1 (OR: 0.27, 95% CI: 0.08-0.86, p = 0.027) were protective factors. These results were accompanied with an area under the curve ROC of 0.86 (95% CI AUC: 0.770-0.925).  A sixth multivariate study was also performed to analyze asymptomatic COVID-19 patients and symptomatic patients who did not require hospitalization (Table 4F). The proportion of the Th1 (OR: 0.23, 95% CI: 0.05-0.96, p = 0.045) immune response against the SARS-CoV-2 infection was a significant and independent protective factor for the development of symptoms with an area under the curve ROC of 0.679 (95% CI AUC: 0.557-0.815).
The final and seventh analysis studied which parameters were associated with asymptomatic and severe forms of the disease (Table 4G and Figure 8). The proportion of activated CD8+ T cells (OR: 6.67, 95% CI: 1.05-45.6, p = 0.044) and the total proportion of %Th1 immune response (OR: 0.09, 95% CI: 0.01-0.63, p = 0.015) resulted in a significant and independent risk and protective factor, respectively, with an area under the curve ROC of 0.873 (95% CI AUC: 0.714-0.922).
A sixth multivariate study was also performed to analyze asymptomatic COVID-19 patients and symptomatic patients who did not require hospitalization (Table 4F). The proportion of the Th1 (OR: 0.23, 95% CI: 0.05-0.96, p = 0.045) immune response against the SARS-CoV-2 infection was a significant and independent protective factor for the development of symptoms with an area under the curve ROC of 0.679 (95% CI AUC: 0.557-0.815).
The final and seventh analysis studied which parameters were associated with asymptomatic and severe forms of the disease (Table 4G and Figure 8). The proportion of activated CD8+ T cells (OR: 6.67, 95% CI: 1.05-45.6, p = 0.044) and the total proportion of %Th1 immune response (OR: 0.09, 95% CI: 0.01-0.63, p = 0.015) resulted in a significant and independent risk and protective factor, respectively, with an area under the curve ROC of 0.873 (95% CI AUC: 0.714-0.922).

Discussion
COVID-19 is a condition that shows a well-characterized spectrum of clinical profiles, going from asymptomatic patients to those who develop life-threatening complications, even death [41]. The latter patients are distinguished by showing alterations in their immunological profile, lymphopenia, neutrophilia or eosinophilia standing out among these [42][43][44].
As has occurred in other infectious diseases, the immune response mounted around the disease is determinant for its outcome. The presence of an effective and well-controlled adaptive immune response is sufficient for the clearance of a mild COVID-19 [45]. However, an exacerbated and persistent adaptive immune response identified by activated and exhausted phenotypes of cytotoxic T-cells with an impairment of the Th response has been associated with severe forms of the disease [32,46].
Based on a comprehensive immune profile created in this present study and the results obtained, we have been able to demonstrate that the early immune response in COVID-19 patients who require hospital admission differs from that developed by patients who do not need to be hospitalized. Interestingly, non-hospitalized COVID-19 patients established an early, effective and robust Th1 response, which behaves as an independent protective factor in the multivariate analysis of factors associated with an unfavorable disease evolution. These facts suggest a similar scenario between COVID-19 and other infective diseases like leishmaniasis, leprosy or HIV infection where the early immune response based on Th1 cells is associated with better outcomes [9,[47][48][49].
Although COVID-19 is a condition that affects a wide range of ages, from childhood to old age, the most serious forms mainly affect the elderly [50][51][52]. Our results are in consonance with that fact; hospitalized infected patients were older than those patients who did not require hospital care. Likewise, the distribution of severity by sex revealed that male gender presented higher rates of hospitalization compared to females. As the severity increases, the proportion of men is higher in each group, and the biggest differences being found are in deceased patients [6,53].
In our cohort, the elevation of biomarkers associated with inflammation, such as LDH, CRP and DD, were associated with higher rates of hospitalization, confirming the relevance of inflammation in the pathophysiology and prognosis of the disease, as previously reported [54].
The T-cell profile in the early stages of the infection was different in those patients who later evolved to more complex forms of the disease and required hospital treatment. While patients who did not require hospitalization showed an immune status similar to that of healthy controls, hospitalized patients presented global lymphopenia, this being in line with previous studies where lymphocyte count had been proposed as a predictor of severity [42,55]. Likewise, these patients presented other important alterations, the phenotype and proportion of Th1 and CD8+ T-cells compartments being especially affected.
From the analysis of the T-cell profile observed in the different clinical variations, we can consider that the evolution of COVID-19 depends on the type of initial Th response. Establishment of a robust Th1 response makes it possible to effectively control viral clearance and avoid the development of complex symptoms: good prognosis COVID-19 patients showed a potent Th1 immune response compared to critically ill patients. These results are in line with other publications where the presence of higher proportions of Th1 cells was associated with mild forms of the disease [56]. The robust Th1 immune response is not only determined by the total proportion of Th1 or senescent cells. The proportion of quiescent Th1 cells could be a marker of the turnover associated with the activation and exhaustion of Th1 immunity.
A work conducted by Salehi Khesht et al. that studied the responses to SARS-CoV-2 with different immunological profiles of T-cells found a positive correlation between the severity of the disease and the Th1 response [57], which could contradict our results. However, Salehi Khesht et al. did not carry out a phenotypic study of the lymphocytic population and their study also did not indicate at what point in time of the infection the immune profiles were evaluated (acute or convalescent), so it is not possible to assess whether they mediated a rapid action against the infection or if it occurred. Furthermore, the increase in Th1 could not only be ineffective, but it also induces a polyclonal activation with the consequent pro-inflammatory scenario and severe forms of the disease [58].
The cytokines made by Th1, mainly interferon-gamma, are essential to mediate cellmediated immunity against viruses and intracellular pathogens [59]. It has been demonstrated in our work that a global Th1 immunity is a protective factor for the development of severe forms of the disease in which hospitalization requirements are needed. A weak or absent Th1 response would imply poor ability of these pathogens to slow the disease progression [10]. When this scenario arises, alternative responses that may be essentially mediated by Th2 and Th17 are developed. In the case of COVID-19, these responses may end up being inadequate and may contribute to the immunopathology of the disease with the production of cytokines and the recruitment of immune cells to the lung [60,61].
The Th2 responses against infection mainly based on the production of antibodies [62] may not be efficient enough to eliminate the infection, since the scope of antibodies at the intracellular level is minimal [63]. Our results have shown how a prominent Th2 response results in severe forms of the disease. This fact confirms the findings in our previous work, where we described that COVID-19 patients who eventually died had established a potent Th2 response as a compensatory mechanism for an impaired Th1 response [32].
The Th17 response has been associated with autoimmune and lung inflammation [64]. Cells of the Th17 lineage present both pathogenic and protective functions in COVID-19: IL-17 recruits innate immune cells in the lungs to eliminate infection, but on the contrary, uncontrolled secretion of cytokines (IL-23/IL-17 axis) could aggravate the pathology, contributing to the cytokine released syndrome (CRS) [65,66]. We have observed a positive correlation of Th17 immunity and disease severity quantified as quiescent and senescent Th17 cells in our cohort. These results are in line with some publications, where the increase of Th17 cell activity has been observed in hospitalized COVID-19 patients, quantified by the production of IL-21 and IL-22 [27,67]. Sarmiento-Monroy et al. suggest that since cytokines released by Th17 in SARS-CoV-2 infection are hazardous in the context of CRS and ARDS, the usage of biological therapies targeting IL-23/IL-17 axis could be potentially beneficial in those scenarios [68].
We have found a negative correlation in the analysis of the absolute number of CD8+ T-cells according to disease progression. This phenomenon is analog to what occurs with CD4+ T-cells in HIV infection, where an adaptive immune response based on an efficient cell-mediated immunity makes it possible to overcome the disease without complications.
Differences were not found when both the CD4+ and CD8+ memory cells were analyzed, comparing asymptomatic to the different grades of prognosis. The instability of the proportion of the memory compartment between the different disease groups suggests long-term protection [69,70].
As many other authors have reported [42,71,72], global lymphopenia, including CD4+ and CD8+ T cells, reflects the depletion and deterioration of the immune system [73,74]. As our results have shown, CD8+ activation could play an important role in the pathogenesis link to a dysregulation of the immune system. The persistent CD8+ activation in severe patients could be translated as the last-ditch effort of the immune system to clear the infection, since the global Th1 response is absent or ineffective when associated with a pathogenic Th17 response, contrary to what has been observed in asymptomatic patients.
Since the Th1 response seems to be responsible for the correct clearance of the infection, new therapeutic strategies in this disease should be aimed at enhancing this type of response. Recent publications have shown that COVID-19 vaccines induce a bias towards Th1 immune response similar to that which we have observed in the natural infection of asymptomatic patients [75][76][77]. Likewise, the development of new vaccines and the selection of adjuvants and delivery systems should not only be oriented towards the production of antibodies but also towards ensuring that robust Th1 immunity is achieved [78][79][80][81].
This study has several limitations. The main limitation is the size of the cohort and although it is not negligible, some groups are still small when stratified according to severity. Due to the high degree of dispersion of the results, the univariate and multivariate evaluation could not be performed with raw data. Therefore, each variable needed to be transformed into ranges using a frequency analysis histogram. In the same way, the number of controls that have never had contact with viral proteins (healthy blood donors) is limited, due to the difficulties in recruiting them during the pandemic and subsequently due to the fact that the vast majority of blood donors in our environment are vaccinated against SARS-CoV-2.

Conclusions
The patients infected by SARS-CoV-2 who have a better evolution are those who establish an initial robust Th1 response against the virus, so that Th2-type responses would be associated with a more complex evolution. Thus, the determination of the type of response which predominates at the beginning of the infection could be used as a tool to discriminate patients who really require hospitalization from those who do not.
New therapies and vaccines should be based on Th1 cellular immune responses stimulation, which could rule out the development of severe forms of COVID-19-like infections.