Zinc-based Nutritional Immunity to Lower Inflammation, Viral Load and COVID-19 Mortality During SARS-CoV-2 Infection.
Phase: PHASE4
Conditions: Zinc Deficiency, Sars-CoV-2 Infection
Sponsor: Parc de Salut Mar

Zinc-based 
nutritional 
immunity 
to 
lower 
inflammation, viral load and COVID-19 mortality 
during SARS-CoV-2 infection. 
 
 
PRINCIPAL INVESTIGATOR COORDINATOR: Robert Güerri-Fernández M.D. Ph.D.  & 
Silvia Gómez-Zorrilla M.D.Ph.D. 
PRINCIPAL INVESTIGATOR SUBPROJECT 1: Rubén Vicente Ph.D. 
 
 
Version: Version 1.0 (29 march 2021) 
 
 
SPONSOR: Consorci Mar Parc de Salut de Barcelona, con domicilio en Passeig Marítim 
25-29, 08003 de Barcelona. C.I.F S-080047 
 
Principal Investigator: Robert Güerri-Fernández M.D. Ph.D. 
Edificio Hospital del Mar. Servicio de Enfermedades Infecciosas. Paseo Marítimo 25-29, 
08003, Barcelona 
Phone number: +34 93 248 3468 / +3493 248 3251 
Fax: +34 93 248 3257 
E-mail: rguerri@psmar.cat 
 
 
 
 
 

ABSTRACT: 
 
Background: The essential micronutrient zinc balances immune responses to infections 
and additionally directly inhibits some viruses. We have recently shown a robust 
correlation between serum zinc levels (SZL)and COVID-19 outcome in patients and a 
direct effect of zinc levels on SARS-CoV-2 expansion in cell culture.These results suggest 
that SZL might represent an important risk factor for COVID-19 severity whose 
adjustment would constitute an early and cost-effective therapeutic intervention. 
Objectives: To explore the therapeutic benefit of zinc supplementation for COVID-19 
patients and to determine the cellular and molecular basis of the effect of zinc levels on 
SARS-CoV-2 infections. 
Methodology: A randomized clinical trial supplementing COVID-19 patients with zinc 
will be carried out and viral loads, inflammatory and novel zinc-related clinical markers 
and SARS-CoV-2-specific T- and B-cell Responses monitored. Humanized-ACE2-mice 
models will be used to address causal-effect associations between zinc levels and SARS-
CoV-2 infection. Infections of human Calu-3 cells will be used to decipher the direct 
antiviral action of zinc on SARS-CoV-2 life cycle. 
Expected results: We will generate a clear insight into zinc functioning in COVID-19 
patients that might provide a therapeutic guideline immediately applicable to reduce 
the severity of SARS-CoV-2 pathogenicity and possibly other virus infections. 
 
 
LAY SUMMARY: 
 

Infections with SARS-CoV-2 result in a systemic disease with a variety of outcomes, from 
no symptoms to severe and diverse pathologies. Therefore, it is important to identify risk 
factors determining COVID-19 severity, especially if those factors might be adjusted, 
allowing early and effective therapeutic interventions. 
Zinc is a trace element essential for human health. Zinc deficiency is common in old 
adults, vegetarians and patients with chronic inflammatory diseases. This condition 
causes immune dysfunction leading to increased risk of inflammatory and infectious 
diseases, including acquired immune deficiency syndrome, measles, malaria, 
tuberculosis, and pneumonia. Besides, zinc has a direct antiviral activity against specific 
viruses like rhinovirus, HCV, herpes simplex virus. In this scenario, it has been shown 
that zinc supplementation has benefits on the recurrence and persistence of acute and 
chronic viral infections like common cold or HCV, HBV. Moreover, our team has recently 
done an observational study with 249 COVID-19 patients that showed how COVID-19 
patients with lower plasma zinc content had worse prognosis, increased time of 
hospitalization and mortality. 
Therefore, the main aim of the project is to explore the therapeutic benefit of zinc 
supplementation for COVID-19 patients and to determine the cellular and molecular 
basis of the effect of Zn levels on SARS CoV-2 infections. For that purpose we will run a 
clinical trial supplementing with zinc COVID-19 patients. Moreover, we will carry out 
experiments to understand the association between zinc nutritional status and SARS- 
Cov-2 infection progression in cellular and animal models. 
 

Given the current knowledge about zinc supplementation toxicity and dosage, we expect 
that recommendations derived from this study will be rapidly applied by physicians and 
public health decision makers. The results of these studies will be used as a guideline to 
administer zinc supplements in COVID-19 patients in order to reduce disease severity 
and mortality. Moreover, our experiments will clarify whether zinc supplementation as a 
prophylaxis strategy is useful to protect the population at risk of zinc deficiency, more 
than 20% worldwide. 
Finally, considering the new knowledge that this project will generate about the role of 
zinc in immune responses and viral expansion, we expect that our results will help 
researchers and physicians to design novel strategies to boost specific immune cell 
subpopulations against SARS-CoV2 infection. Thus, this knowledge could be used long- 
term for designing medicines against SARS-CoV-2 and other viral infections. 
 
• 
HYPOTHESIS AND DESCRIPTION OF THE PROJECT OBJECTIVES 
 
We hypothesize that low zinc nutritional status has an impact on COVID-19 progression and that 
adjustment of this status might constitute an early therapeutic intervention point by balancing 
immune responses and acting as an antiviral agent. This hypothesis is supported by the following 
observations: 
-Zinc nutritional status impacts on innate and adaptive immunity and influences inflammatory 
markers(4). 
-Zinc has a direct antiviral activity against specific viruses like rhinovirus, HCV, SARS-CoV(16). 
 
-Zinc supplementation has benefits on the recurrence and persistence of acute and chronic viral 
infections like common cold, or HCV, HBV (2,6) and reconstitutes the production of interferon- 
alpha from elderly persons(16). 

-Our recent data in a cohort of 249 COVID-19 patients show that COVID-19 patients with lower 
plasma zinc content have worse prognosis, increased time of hospitalization and mortality and 
that Zn and IL-6 levels are negatively correlated (13). However, the cause-effect association 
between nutritional zinc status and COVID-19 inflammatory response remains 
unproven, 
 
-Our recent data in cell culture studies show that low zinc levels correlate with higher SARS-CoV- 2 
expansion(13) . 
 
 
The main aim of the project is to explore the therapeutic benefit of Zn supplementation for 
COVID-19 patients and to determine the cellular and molecular basis of the effect of Zn levels on 
SARS CoV-2 infections. To address these questions, we propose the following aims: 
 
 
• To study the impact of zinc supplementation on COVID-19 progression. There are two 
specific objectives: 
• 
To carry out the clinical trial with COVID-19 patients (Subproject 1): 
 
We will run a randomized clinical trial with 60 COVID-19 patients admitted at Hospital del Mar 
with a control and a zinc supplementation group. 
 
 
• 
To study the immune responses of the COVID-19 patients enrolled in the clinical trial 
(Subproject 2): Viral loads, zinc homeostasis markers and T- and B-cell populations in PBMC from 
COVID-19 patients in control and zinc supplemented groups will be determined. Disease 
progression and clinical outcomes will be evaluated. 
 
 
• To establish causal-effect associations between zinc levels and SARS-CoV-2 infections 
(Subproject 3): We will monitor viral expansion and immune responses in SARS-CoV2 infected 

hACE2-mice with different zinc nutritional status (i) to evaluate whether SARS-Cov-2 induces 
changes in SZL and (ii) to study prophylaxis and therapeutic properties of zinc supplementation. 
 
 
• To characterize novel zinc homeostasis reporters upon SARS-CoV2 infection (Subproject 
2) We will characterize the expression of zinc transporters and metallothioneins in PBMC from 
COVID-19 patients and mice infected with SARS-CoV-2. 
 
 
• To decipher the direct antiviral action of Zn on SARS-CoV-2 life cycle steps 
 
(Subproject 2)Infections of human Calu-3 cells will be used to decipher the direct antiviral action 
of Zn on SARS-CoV-2 life cycle steps. Moreover, zinc homeostatic machinery will be evaluated 
upon infection. 
 
 
 
2. 
BACKGROUND AND STATE OF THE ART, INCLUDING RELEVANT BIBLIOGRAPHY 
 
 
Zinc is a trace element essential for human health. Zinc deficiency (ZD) causes impaired 
body growth, neurological disorders, and immunosuppression leading to morbidity and 
increased risk of inflammatory and infectious diseases including acquired immune 
deficiency syndrome, measles, malaria, tuberculosis, and pneumonia (1,2). The human 
body contains 2–3 g of zinc, 0.1% of which are exchanged daily. Due to the lack of major 
storage organs of this mineral, the body needs a continuous nutritional supply. Zinc 
deficiency is considered a major public health problem in developing countries, 
especially in groups with low zinc intake as well as those with inhibited absorption due 
to high levels of phytates and fiber in vegetable-based diets. Besides, old adults, 
vegetarians, and people with gastrointestinal disorders are also considered to be at risk 

for hypozincemia(1, 2). Globally, one third of the world population is at risk of mild ZD 
associated disorders. In the elderly, the incidence ranges from 15 to 31% depending on 
the age and the country of study (3). Remarkably, older adults are the group at higher 
risk for severe symptoms and mortality from COVID-19. In this context, ZD might be one 
of the important underlying factors of COVID-19 heterogeneity. 
 
 
Cellular zinc homeostasis 
 
Zinc is a structural component of up to 10% of the proteome. Moreover, zinc participates as 
a second messenger in cell signalling. Zinc binding proteins and 24 zinc transporters 
work in a coordinated way to tightly regulate cytosolic zinc fluxes. There are two main 
families of zinc transporters, ZnT and Zip, which extrudes and enters zinc in the cytosol 
 
respectively (1). Within each zinc transporter family, the different members vary in 
cellular localization, affinity, selectivity, regulation and tissue expression. The expression 
and function of specific transporters is essential for the function and modulation of 
immune cells of adaptive and innate immunity (4). 
 
 
The concentration of free zinc in the cytosol is not only determined by the activity of all 
these transporters but also by the quenching effects exerted by metallothioneins (MTs), 
abundant cytosolic proteins that quench metal ions, mainly zinc. These proteins, by 
releasing zinc, participate actively in the defense against reactive oxygen species (ROS). 
In humans, there are 4 MTs. Interestingly, the cellular expression of MTs is stimulated by 

increases in cytosolic zinc content. In fact, one of the most reliable tests to measure the 
body zinc content is by measuring MTs expression in leukocytes (5). 
 
 
Zinc and immune cells 
 
Early studies of zinc deficiency showed that zinc is essential for immune system function 
(4). Due to its importance for proper immune function, zinc supplementation and 
fortification programs have been widely carried out for boosting the immune system in 
populations at risk of ZD. Several clinical trials have proven that zinc supplementation is 
effective in certain types of viral and bacterial infections (reviewed in (2,6)). 
 
 
Zinc and inflammation 
 
ZD is known to be associated with proinflammatory responses at infection, showing 
higher ROS production and inflammatory markers (6,7). An imbalance in cytokine 
production by cells of both innate and adaptive immunity has also been reported. 
 
Concretely, ZD is associated with higher levels of IL-6, IL-1beta and TNF-alfa (6). In this 
scenario, zinc supplementation has been shown to decrease the incidence of infection, 
inflammatory cytokines, including IL-6, and oxidative stress markers in elderly 
individuals (7,8). Moreover, zinc supplementation has been shown to balance immune 
responses (9). Remarkably, IL-6 is a key player in COVID-19 patients by triggering severe 
lung damage during SARS-CoV-2 infection. 
 
 

Sustained IL-6 promotes immune-mediated lung damage and macrophage activation 
syndrome (10). Nutritional zinc status and IL-6 are highly interconnected. Thus, IL-6 can 
reduce zinc bioavailability promoting internalization in hepatocytes and the expression 
of zinc chelators (11). On the other hand, zinc decreases IL-6 production via inhibition of 
STAT-3 pathway (12). Our study with COVID-19 patients showed a negative correlation 
between serum zinc levels and IL-6 production (13). Both parameters were robustly 
associated with COVID-19 progression. This data is in agreement by another 
observational study with a smaller cohort (14). However the cause-effect association 
between zinc status and inflammation markers in COVID-19 patients is not yet clear 
given the observational nature of the studies (13,14). 
 
 
Zinc in viral infection 
 
Zinc is an essential trace element for both host and pathogens. In order to grow, 
pathogens require zinc. Thus, zinc is a structural cofactor of viral proteins and certain 
viruses have developed strategies to alter cellular zinc homeostasis on its benefit (15). 
On the other hand, there is extensive evidence that zinc can prevent viral replication and 
lower cellular invasion (eg. Rhinovirus, HCV, SARS-CoV) (reviewed in 6, 16). 
 
Remarkably, our team has shown that low zinc levels favour SARS-CoV2 expansion (13). 
These results might indicate either a direct anti-viral action against SARS-CoV2 or that 
the virus benefits from stress conditions caused by low levels of cellular zinc. Although 
there are several evidences showing that zinc deficiency leads to increased susceptibility 
to certain infections, supplementation of zinc in viral infections has not always been seen 

as beneficial. For example, zinc supplementation in HIV patients does not improve T-cell 
counts or reduces viral load. In contrast, zinc supplementation has been shown to be 
beneficial when administering to HBV and HCV infected patients. Moreover, zinc- based 
nutritional immunity reduces diarrheal episodes and respiratory track diseases 
(reviewed in 6,16). Remarkably, zinc supplementation for the common cold caused by 
rhino- and coronaviruses prevents it and reduces its duration, if the administration starts 
within 24h after the onset of symptoms (reviewed in 6,16). Thus, zinc supplementation 
has been proven beneficial against some viral infections. However, whether this was 
caused by improved local immune response or viral inhibition remains uncertain. 
 
 
IFNs are key antiviral mediators of the host's immune response. Impaired IFN responses 
related to aging have been associated with increased susceptibility during certain viral 
infections. Older adults are more susceptible than younger adults to death resulting from 
viral infections due to a decline in interferons production. Interferons are antiviral agents 
produced by the host upon infection. Importantly, zinc supplementation reconstitutes 
the production of interferon-alpha by leukocytes from elderly persons (17). This is an 
important aspect when considering zinc based nutritional immunity 
 
because SARS-CoV-2 has been shown to antagonize proinflammatory signals during early 
infection, particularly type I IFN (IFN-I) signaling. In fact, errors of immunity that 
diminish IFN-I activity are more commonly detected in patients with severe 
COVID-19 (18). 
 
 
 

Zinc in T lymphocytes activation and exhaustion 
 
Several studies have indicated that T cells have a role both in the early immune response 
against SARS-CoV-2 and that a functional memory is generated (19). In this context, zinc is 
known to affect T lymphocyte maturation, differentiation and cytokine production 
(2,4,7). Activated T cells are known to increase zinc content (20). On the one hand, zinc 
has been shown to be required for the correct TCR signalling by modulating Lck and Zap- 
70 activity (20). Our team has demonstrated that zinc positively potentiates the three 
main signalling pathways, AP-1, NF-kB and NFAT1 (21). In addition we have described 
that the knock down of Zip6, a zinc transporter up regulated early during cell activation, 
alters zinc entry and dramatically impairs activation of Jurkat T cells (21). Moreover, 
recent studies on CD8+ T cell dysfunction during cancer and chronic infections have 
started to elucidate the transcriptional pathways involved in this phenomenon and zinc 
homeostasis has emerged again as a key element. Thus, metallothioneins 1 and 2 levels 
and zinc dependent transcription factors are differentially expressed in exhausted CD8+ 
cells (22,23,24). Nonetheless, there are no studies investigating whether zinc 
supplementation/chelation might directly affect the CD8+ dysfunctional profile and how 
these learnings could be translated into novel therapeutic approaches for these diseases 
including the current COVID-19 pandemic. 
 
Zinc in B lymphocyte function 
 
B lymphocytes are key players for an effective immune response against SARS-CoV2. 
Antibodies against the spike and nucleocapsid proteins of the virus are detectable from 
approximately 6 days after PCR confirmation of infection. A decline of anti-SARS-CoV-2 

serum IgG levels occurs beyond 20 days post-diagnosis and it is still unclear how long 
immunity to SARS-CoV-2 lasts after recovery from infection. Importantly, it has been 
shown that zinc transporters (Zip7 and Zip10) are essential in B cell function. B cells 
activation and plasma cell differentiation depends on zinc signalling (25,26). Nutritional 
zinc status is an important factor determining antibody production given its action on 
both, B-cells and CD4+ T lymphocytes. Thus, ZD has been shown in mouse models to 
lower humoral immune response (26). 
 
 
In summary, there are several cross talks between zinc homeostasis and immune 
function. Moreover zinc has an antiviral activation against certain viruses. All these 
connections together with the association found between low plasma zinc levels and 
COVID-19 severity suggest that zinc supplementation might be an important therapeutic 
tool in COVID-19 patients. A clinical trial based on zinc nutritional immunity together 
with a deeper knowledge of the impact of zinc supplementation in immune cells will be 
useful for the current COVID-19 pandemic and other infection diseases 
 
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Transporters in Zinc Homeostasis and Metabolism. Physiol. Rev. 95: 749–784. 
• Gammoh,NZ, et al.(2017) Zinc in Infection and Inflammation. Nutrients 9: 624. 
 
• Yasuda H, et al.(2016) Infants and elderlies are susceptible to zinc deficiency. Sci Rep 
 
2016;6:21850 

 
• Haase H, et al.(2009) Functional significance of zinc-related signaling pathways in 
immune cells. Annu. Rev. Nutr. 29: 133–52. 
• Hennigar SR, et al.(2016) Metallothionein and Zinc Transporter Expression in 
Circulating Human Blood Cells as Biomarkers of Zinc Status: a Systematic Review. Adv 
Nutr. 7:735-46. 
• Overbeck, S. et al.(2008) Modulating the immune response by oral zinc 
supplementation: a single approach for multiple diseases. Arch. Immunol. Ther. Exp. 
(Warsz). 56: 15–30. 
• Prasad AS et al.(2007) Zinc supplementation decreases incidence of infections in the 
elderly: effect of zinc on generation of cytokines and oxidative stress. Am J Clin Nutr 
2007;85:837–44. 
• Bao B, et al.(2010) Zinc decreases C-reactive protein, lipid peroxidation, and 
inflammatory cytokines in elderly subjects: a potential implication of zinc as an 
atheroprotective agent. Am J Clin Nutr 2010;91:1634–41. 
• Rosenkranz, et al.(2016) Induction of regulatory T cells in Th1-/Th17-driven 
experimental autoimmune encephalomyelitis by zinc administration. J. Nutr. Biochem. 29: 
116–123. 
 
• Beck FW, et al.(1997) Changes in cytokine production and T cell subpopulations in 
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Liuzzi JP, et al.(2005) Interleukin-6 regulates the zinc transporter Zip14 in liver and 
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• 
Liu MJ, et al.(2014) Zinc regulates the acute phase response and serum amyloid A 
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• 
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• Hadjadj J, et al.(2020) Impaired type I interferon activity and inflammatory responses 
in severe COVID-19 patients. Science 369, 718–724. 
• Karlsson AC, et al.(2020) Sci. Immunol. 10.1126/sciimmunol.abe8063. 
 
• 
Yu M. et al.(2011). Regulation of T cell receptor signaling by activation-induced zinc 
influx. J. Exp. Med.208: 775–85. 
 

• 
Colomar-Carando N, et al.(2019). Zip6 transporter is an essential component of the 
lymphocyte activation machinery. J. Immunol 202(2):441-450 
• 
Singer M, et al.(2016). Distinct Gene Module for Dysfunction Uncoupled from 
Activation in Tumor-Infiltrating T Cells. Cell. 166:1500-1511. 
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Doering TA, et al.(2012) Network analysis reveals centrally connected genes and 
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Dominguez CX, et al.(2015) The transcription factors ZEB2 and T-bet cooperate to 
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Exp Med. 212:2041-56. 
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Anzilotti C, et al.(2019). An essential role for the Zn2+ transporter ZIP7 in B cell 
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• 
Hojyo S, et al.(2014) Zinc transporter SLC39A10/ZIP10 controls humoral immunity 
by modulating B-cell receptor signal strength PNAS 111, 11786-11791 
 
 
3. PRELIMINARY RESULTS: 
 
Our preliminary results are shown in the work entitled “Low zinc levels at clinical 
admission associates with poor outcomes in COVID-19” (13) which is the result of a 
tight collaboration between the laboratory of Roberto Güerri (Hospital del Mar) and 
those of Rubén Vicente and Juana Díez (both at DCEXS, UPF). 
This work aimed to focus clinical attention on serum zinc content (SZC) in COVID-19 
patients. Our analysis showed a robust correlation between low SZC with COVID-19 

severity and mortality. The cause is likely to be a combination of immune system 
imbalance and a direct benefit of viral replication. 
 
 
A summary of the results obtained: 
 
 
 
• In our retrospective observational study with 249 COVID-19 patients, 23% of 
the patients had at admission SZC lower than 50µg/dl, the cutoff associated 
with ZD and development of clinical signs. 
• Our study showed that low SZC correlates with higher IL-6 and C-reactive 
protein at admission, higher peak IL-6 during the episode. 
• 
 
We detected in the low SZC group that subjects needed higher time to 
recover (25 days) versus the control group (8 days). In the same direction, the 
mortality in low SZC patients was higher (21%) than in the control group 
(5%) with normal zinc concentration. 
• SARS-CoV2 Infections in cell culture demonstrated that low zinc levels favors 
SARS-CoV2 viral expansion in the infected cells. 
 
 
These results highlighted the urgent need to start clinical trials supplementing with zinc 
low SZC patients at admission to reestablish zinc homeostasis in order to study the 
cause-effect association between zinc nutritional status and COVID-19 progression. We 
expect with this new project that zinc supplementation in low SZC COVID-19 patients will 

balance what has been called the cytokine storm caused by SARS-CoV-2 and as a 
consequence will reduce COVID-19 severity and mortality. 
 
METHODOLOGY: 
 
 
 
The methodology is divided into objectives, subobjectives and tasks. Expected outcomes 
(EO), deliverables (D) and limitations are described for each task. 
 
 
• 
To study the impact of zinc supplementation on COVID-19 progression. 
 
 
 
• 
To carry out the clinical trial with COVID-19 patients (Subproject 
1) Clinical trial objectives 
Primary objective 
 
 
To evaluate the effectiveness of oral zinc supplementation   (83.91 mg zinc acetate) 
 
Zinc+NM QD  (80mg Zn2+)  QD  as compared to placebo  as compared to control group 
 
without zinc supplementation to reduce IL-6 increase in the course of SARS-CoV-2 
infection, to decrease days to clinical recovery and to decrease the percentage of people 
with COVID-19 who have disease progression by Week 2, defined as inpatients, 
worsening of ≥1 level on the WHO 9-point ordinal scale 
 
Secondary Objectives 
 
 

To evaluate the effects of zinc supplementation QD on fever, dyspnea, diarrhea, and 
clinical parameters of disease, and time to worsening clinical status. 
 
Study Endpoints 
 
 
Primary Endpoints 
 
• Number of days since admissions to clinical recovery 
 
 
• Change in IL-6 levels from baseline to Week 2. 
 
 
• Percentage of patients with disease progression by Week 2 based on WHO 9-point 
ordinal scale 
 
Secondary Endpoints 
 
 
• Change in Covid-19 signs/symptoms: dyspnea, diarrhea, temperature, neutrophil 
count... at Week 2 
 
Safety Endpoints 
 
 
• Incidence of AEs and serious adverse events 
 
 
 
 
 
Study Design 
 
 
This is a randomized, open-label clinical study evaluating the effectiveness of zinc 
supplementation as compared to standard of care on need disease severity and on IL-6 

levels in people with COVID-19. Subject eligibility will be based on inclusion and 
exclusion criteria 
 
 
 
Population of study: Sixty eligible adult subjects at one clinical site in Spain (Hospital del 
Mar) will be randomized in a 1:1 ratio to: 
 
• Zinc acetate 80mg QD 83.91 mg zinc acetate (Zinc+NM) QD + standard of care for 2 
 
weeks 
 
 
• Standard of care for 2 weeks 
 
 
Study phases (4 weeks): 
 
 
• Screening + Randomization + Baseline Visit (Visit 1, same day as or up to 1 day before 
Baseline Visit) 
 
• Treatment (Day 1 to Day 14) Starting on Day 1 (Baseline/Randomization), Randomized 
subjects will receive 80mg zinc for 2 weeks. Day 7 for clinical state. Day 14 follow up 
visit. 
 
• Follow up phase : Day 28 for clinical state. 
 
 
Inclusion Criteria 
 
 
To be eligible to participate in this study, all of the following criteria must apply: 
 
 
• Subject is ≥18 years of age. 

 
 
• Male or female. 
 
• Subject with diagnosis of COVID-19 based on + RNA or IgM test or compatible clinical 
presentation. 
 
• Ability to take oral zinc supplementation. 
 
 
• Subject or surrogate decision maker is capable of understanding and willing to sign the 
informed consent form. 
 
Exclusion Criteria and withdrawals are detailed in the document sent to the Ethics 
 
Committee. 
 
 
Subjects are not permitted to enroll in the study if any of the following criteria apply: 
 
 
• Female subjects who are pregnant 
 
• Female subjects who are breast-feeding. 
 
• Difficulty or inability to take oral medication. 
 
• 
Subject is enrolled in another randomized clinical trial in the last 3 months. 
 
• Life expectancy less than 72 hours at hospital admission. 
 
• 
Conditions that in the opinion of the investigator may interfere with the subject’s 
 
ability to comply with this protocol (e.g drug abuse, psychiatric disorders). 
 
 
 
 
Task 1.1.1 Visits and sample collection 
 

 
1.- Visit 1. Screening. Randomization. Baseline. Phase / Visit 0 or 1 ( Day 1 or up to 1 day 
Prior to Baseline/Randomization; In Person) 
 
Visit performed at admission (+1). Written informed consent obtained. Medical history. 
Vital signs assignment. Clinical labs (CBC, CMP, CRP, ferritin, D-dimer,IL-6, zinc) 
 
2.-Visit 2 / Week 1 (Day 7 ± 2 days) 
 
 
A complete record of AEs and concomitant therapies will be taken into account. 3.-
Visit 3 / Week 2 (Day 14 ± 2 days), End of Treatment 
 
A complete vital sign assessment (sitting blood pressure, heart rate, respiratory rate, O2 
sat, and oral temperature) review and record AEs and concomitant therapies, clinical 
labs (stated above) and PBMCs. Collect blood samples for future studies. 
 
4.-Visit 4 / End of Study (Day 28 ± 4 days) 
 
 
Clinical labs (stated above) and PBMCs. Collect blood samples for future studies. 
 
 
Task 1.1.2 Assessment of Efficacy 
Outcomes measured. 
• Clinical recovery 
 
 
• 
 Defined: Sat O2 (0.21) >94% or to baseline Sat O2 in case of chronic 
respiratory 

• 
Temperature <37C 
 
• 
Heart Rate <90 bpm & Systolic blood pressure >100 
 
• 
Oral tolerance 
 
• 
Baseline consciousness 
 
 
• Assessment of Covid-19 Disease Severity as per the WHO 9-point ordinal scale 
Ordinal scale, 9 points 
• IL-6 levels 
 
 
EO: We expect to prove the effectiveness of zinc supplementation to reduce COVID-19 
severity and mortality 
 
D1: Guideline for zinc-based nutritional supplementation to treat COVID-19 patients 
admitted in hospitals 
 
 
 
 
 
 
Statistical analysis 
 
 
Randomization: Subjects will be randomized in a 1:1 ratio to: 
 
 
• 
 83.91 mg zinc acetate (Zinc+NM) QD + standard of care for 2 weeks 
 
• 
 Standard of care for 2 weeks 
 
Determination of sample Size: Sample size calculations are aimed at comparisons of 
days to clinical recovery between zinc supplementation and standard of care alone. 
Assuming alpha = 0.05 and a 10% dropout rate, randomization of 60 subjects will 

provide 80% power to detect a difference in time to recovery between arms of 3 days 
assuming a SD of 4 days. This sample size will also provide at least 80% power to detect 
a change in percentage of people with clinical progression of 5% based on an estimated 
 
 
 
 
 
 
 
clinical progression rate of 20% in the standard of care arm. 
 
Analysis Populations: The analysis population for efficacy will be per protocol which 
includes all randomized subjects who complete 2 weeks of therapy and/or return for 
Week 2 visit. All safety analyses will include all randomized subjects who are 
 
 
 
 
administrated at least 1 packet of the investigational product. 
 
Baseline Characteristics: Demographics and Baseline characteristics will be summarized 
 
descriptively by group and overall using per protocol population. The following Baseline 
 
 
characteristics will be summarized: sex, race, age, comorbilities, ethnicity 
 
 
 
Clinical Laboratory Assessments: Blood chemistry, hematology parameters will be 
summarized at Baseline as well as visit 3 and 4. Laboratory values at Baseline and 
subsequent changes in laboratory values will be summarized descriptively overall and 
 
 
 
 
by therapy group. 
 
 
 
Vital signs: Vital sign measurements will be summarized at Baseline, Week 2 and Week 
4. In addition, changes from Baseline to Week 2 or 4 will be summarized. For each 
summary the number, mean, median, standard deviation, minimum, and maximum 
 
 
 
 

values will be presented overall and by therapy group. 
 
 
 
Safety Analyses: All safety analyses will be based on the Safety Population, comprising 
all subjects who are randomized to the therapy group and subsequently receive at least 
one packet of investigational product. Safety assessments will be analyzed by frequency 
of events/abnormalities for categorical values or summarized using descriptive statistics 
 
 
 
 
 
(mean, standard deviation, median, range, and number of observations). 
 
 
 
Adverse events: Adverse events will be coded using the Medical Dictionary for 
Regulatory Activities (MedDRA) coding dictionary. Adverse events will be defined as any 
event with a start date occurring on or after initiation of therapy on Day 1 or, if pre- 
existing, worsening after initiation of therapy on Day 1. The incidence of AEs will be 
 
 
 
 
 
summarized by body system and MedDRA preferred term, overall and by therapy group. 
 
If a subject reports the same AE more than once, then that subject will only be counted 
 
once for the summary of that AE, using the most severe intensity. 
 
 
 
Handling of Dropouts or Missing Data : Dropouts or participants missing data for the 
 
primary endpoints will be excluded from the analysis. 
 
 
 
Limitation: We might find no benefit in COVID-19 patients supplemented with zinc. That 
would mean that the low zinc levels detected in COVID-19 patients with worse prognosis 
are a consequence of the inflammatory status. The results will be also very helpful for 
physicians worldwide. 

 
 
• 
To study the immune responses of the COVID-19 patients enrolled in the 
clinical trial (Subproject 2) 
 
 
Task 1.2.1 Assessment of B-cell responses in COVID-19 patients 
 
 
 
We will use sera samples from COVID-19 patients enrolled in the clinical trial described 
in objective 1.1 in both groups. We will measure antibody production against SARS- CoV2 
virus at admission, 2 weeks and 28 days post admission. We will carry out ELISA 
experiments to measure IgM, IgA, IgG1 and IgG2a immunoglobulins against recombinant 
SARS-CoV2 spike protein. 
 
 
Task 1.2.2 Assessment of antiviral T-cell responses in COVID-19 patients 
 
 
We will use PBMC from COVID-19 patients enrolled in the clinical trial described in 
objective 1.1 in both groups. We will evaluate T cell responses using intracellular staining 
and flow cytometry. Subtype population (CD3, CD4, CD8), activation degree (IL- 2, INFg, 
perforin), exhaustion (PD-1, Tim-3) and memory (CD45RO and CD62L) markers will be 
evaluated using panels of tagged antibodies. We will stimulate T-cells using a commercial 
pool of overlapping spike peptides of SARS-CoV2. We will study activation degree and 
exhaustion at admission and at 2 weeks. We will evaluate memory at 28 days post 
admission. 
 
 

EO: We expect to know whether zinc supplements might strengthen humoral and cellular 
adaptive immunity in COVID-19 patients. This will be of special interest for 
immunologists and pharma companies 
 
 
D2: Publication describing the results obtained in the clinical trial with COVID-19 
patients and the influence of zinc supplementation in their immune response 
 
 
 
 
2. 
To establish causal-effect associations between Zn levels and SARS-CoV-2 
infections (Subproject 3) 
 
 
Task 2.1 To monitor SARS-CoV-2 infection progression in susceptible mice with 
different Zn supplementations. 
 
We will use as an infection model our own colony of transgenic mice expressing human 
ACE2 (K18-hACE2). Animals will be fed with different supplemented zinc diets (Research 
diets Inc, or similar). Groups of mice (n=21) will be fed with standard diet, zinc deficiency 
(ZD) diet (10mg Zn/kg), adequate (AD) diet (30mg Zn/kg) and supplemented (SD) diet 
(150 mg Zn/kg) for 30d before infection to the end of the experiments. Zn levels in the 
serum of mice will be determined before challenge with infectious virus. Mice will be 
anesthetized and intranasally challenged with a lethal dose of a Spanish SARS-CoV-2 
D614G(S) isolate and followed for 3 weeks. 

Evaluation will be performed by assessing the mice’s clinical health score (appearance, 
mobility and alertness, body weight and temperature) and mortality rates after being 
challenged with SARS-CoV-2 following standardized guidelines. Mice will be bled at 
different times post-infection and the viremia will also be compared between different 
treatments. 
 
 
Determination of sample size 
 
 
 
Group size was calculated according to the equation in (PMID: 24250214) for 
quantitative endpoint data in order to detect a significant 45% change in survival and 
considering a 5% of significance level (P<0.05) and 80% power of the analyses. 
 
 
Task 2.2 To study impact of zinc nutritional status on adaptive immune responses 
against SARS-CoV2 
 
Groups of K18-hACE2 mice (n=30) will be fed and challenged as in task 2.1. Ten 
mice/group will be anesthetized and sacrificed 7, 15, and 21 d.pi., and spleen and PBMC 
will be used. T-cell responses will be evaluated using intracellular staining and flow 
cytometry. Subtype population ( CD4, CD8, ...), activation degree (IL-2, INFg, perforin), 
exhaustion (PD-1, Tim-3) and memory (CD45R0, CD44m cd103a, CD62L) markers will be 
evaluated using panels of tagged antibodies. We will stimulate T-cells using a commercial 
pool of overlapping spike peptides of SARS-CoV2. Antibodies against SARS- CoV-2 elicited 
in sacrificed mice will be also evaluated by ELISA to compare humoral immune response. 

 
 
Sample size 
 
 
 
Group size was calculated using Lehr’s formula to have a power of 80%, considering a 
5% level of significance, and a difference of 13 MFI in the CD4+ T cells between the two 
group means and assuming a combined standard deviation of 10 MFI (PMID: 18341999). 
 
 
EO: We expect to answer the question whether zinc nutritional status before SARS-CoV2 
infection determines COVID-19 progression. Moreover, we expect to understand the 
correlation between serum zinc levels and disease progression. 
 
 
D3: Scientific communication addressed to physicians and public health decision makers 
describing the potential benefits of using zinc supplementation as a prophylactic strategy 
against severe COVID-19. 
 
 
3 To characterize novel zinc homeostasis reporters upon SARS-CoV2 infection 
(Subproject 2) We will run RT-PCR for the 14 Zip and 10 ZnT transporters and for MT1 
and MT2. The expression analysis will be correlated to serum zinc levels measured. 
 
 
Task 3.1 Characterization of machinery for cellular zinc handling in PBMCs from 
COVID- 19 patients 

We will study the expression in PBMCs from COVID-19 patients enrolled in the clinical 
trial (objective 1.1) in both groups at admission and at 2 weeks. 
Task 3.2 Characterization of machinery for cellular zinc handling in PBMCs from 
SARS- CoV-2 infected mice. from mice 
We will study the expression in PBMCs from SARS-CoV-2 infected mice at 7 days post 
infection (objective 2.1) in the three groups of mice (deficient, adequate and 
supplemented diet). 
 
 
EO: Zinc plasma levels are very dependent on inflammatory signalling. We expect to find 
certain genes differentially regulated depending on the nutritional zinc status and 
COVID-19 progression. 
D4: A novel and reliable diagnostic tool in the hospitals to check zinc nutritional status 
and define therapeutic strategies with COVID-19 patients and for other infectious 
diseases. 
 
 
 
 
4. To decipher the direct antiviral action of Zn on SARS-CoV-2 life cycle steps 
 
(Subproject 2 UPF team) 
 
 
 
In both tasks we will use infections of SARS-CoV-2 in the human lung Calu-3 cell lines 
already established in our group. Cells will be infected at high mois to mimic synchronize 
infections. 
 
 

Task 4.1 To determine virus-induced changes in the zinc homeostatic machinery 
and in the intracellular zinc levels. 
 
 
At time points after infections representing early, middle and late events in the infection 
cycle we will first measure zinc levels at the cytosol (pCDNA Zif-CY1)and the 
endoplasmic reticulum (pcDNA-ER-Zap-CY1), the site of SARS-CoV-2 replication. For this, 
we will transfect, fix cells and measure by fluorescence microscopy. Second, we will test 
whether SARS-CoV-2 infection induces changes in the expression of a set of host genes 
related to Zn homeostasis described in objective 3. 
 
 
Task 4.2. To decipher the direct Zn antiviral action on the different SARS-CoV-2 life 
cycle. 
 
 
We will carry out infection kinetics to study whether Zn levels affect early or late steps 
during cell cycle following protocol previously described (13). Moreover, to study 
specific effects in entry, we will use Pseudotyped GFP rSARS-CoV-2 Spike and to study 
effects in assembly/exit defects we will compare differences in intracellular/extracellular 
of SARS-CoV-2 RNA. Once the precise step of the cycle 
 
affected by Zn levels have been detected, we will address the molecular mechanism 
involved. 
 
 

EO: We expect to comprehensively determine the molecular mechanisms involved in the 
direct antiviral potential of zinc against SARS-CoV-2 
 
 
D5. Scientific communication on the effect of Zn on the SARS-CoV-2 life cycle and its 
antiviral activity.