**2. Methods**

Just until recently, India has had the second or third highest COVID-19 infection rate in the world. However, the mortality rate has been comparatively low, possibly due to a relatively younger average age of the general population. There is another current hypothesis that a national Bacillus Calmette-Guérin (BCG) vaccination program in countries seems to be associated with reduced mortality from COVID-19 and India has such a program [26]. This purely observational study to establish dosage and tolerance of prolonged vitamin and mineral supplementation was carried out at a private clinic located in a small town in the District of Nawan Shahr near the historic city of Rahon in North India (Kapoor Singh Canadian Hospital). There were 7500 inhabitants in the town and a total of five COVID-19 cases (4 male and 1 female) between March (when the epidemic began in India) and December 2020. Interestingly, there were no mortalities among these patients who were quarantined in a neighboring city and one of the main treatments current in India at this time was the malarial medication, chloroquine, which also displays anti-inflammatory activity [27]. There was a total state-wide lockdown between March and mid-June 2020. The clinic decided to re-open in July 2020 following the lockdown in the absence of any available coronavirus vaccine. During the second part of the study (January–March 22, 2021), approximately 50 coronavirus cases were recorded in this small town & adjoining village with a total of 1 or 2 deaths as a result of COVID-19 infection. At this stage, treatment at civil hospitals included steroids and antibiotics such as azithromycin for secondary infections [28]. Testing was also more widely available during this second surge, which started in March, peaked in May, and started to subside in October 2021.

The clinic that took part in this trial employed a total of 15 staff members; 9 men and 6 women. They included 2 doctors (one male, one female), 3 nurses, 2 laboratory technicians, 1 security guard, 1 cleaner and 6 general maintenance staff. All participants consented to take part in the study. Although, all 15 staff members participated in the first part of the study, one general staff member (a clinic clerk) dropped out of the second part of the study as a result of being vaccinated and thereby reduced the test group to 14. Although, the medical staff (7) were aware of the potential benefits

#### *COVID-19 Prevention through Vitamin C, D, and Zinc Supplementation: A Small Clinical… DOI: http://dx.doi.org/10.5772/intechopen.103963*

of supplementation, the non-medical staff members (7/8) were not aware of the potential health benefits. However, they were informed that the supplements were not harmful in any way. In addition, all the non-medical staff was not equally exposed to patients as the medical staff. Each staff member took part voluntarily in the first trial that was initiated on July 1, 2020 and extended to December 31, 2020 and in the second trial that was initiated on January 1 and extended to March 22, 2021. It is unlikely that the townspeople were taking oral dietary supplements of any kind since they are not that popular in India and provided the background population for this study. The background population establishes the presence of active coronavirus cases in the community and forms a basis for comparison.

Vitamin, mineral, and amino acid supplementation is not an uncommon practice at European health clinics. For example, specific combinations of vitamins and minerals may be used to promote general good health. One such formula prescribed at a German vegetarian health clinic (Schlosspark Klink) included CoQ-10 (which stimulates ATP production), vitamin D3, and zinc. Supplementary protein pills were used regularly to complement the diet and boost the body's overall metabolism in patients. Moreover, during the SARS-CoV-2 pandemic, they routinely recommended 20,000 IU per week of vitamin D3 (spring to fall) and 40,000 IU per week of vitamin D3 (winter months) as a preventive measure to their guests based on laboratory blood tests, even to those who regularly included meat and fish in their diet. Vitamin D was measured in blood samples from patients as *25-hydroxycholecalciferol* and a minimum concentration of *55 ng/ml* was recommended by the clinic doctors (whereas the sufficiency scale range is 20–70 ng/ml); although anything below this value was not deemed as representing a deficiency, it was judged as being too low for effective COVID-19 prevention.

Thus, in addition to standard precautionary measures adopted universally during the pandemic, a careful selection of vitamin and mineral supplements was made to help protect the staff at the Indian clinic participating in this particular study from coronavirus infection. The supplements selected for the staff included a combined daily dose of vitamin C (500 mg) and zinc (20 mg) in tablet form [Indian Drugs & Pharmaceutical Co.] plus a weekly dose of vitamin D3 (60,000 IU) capsules [Dr Morpen; Cipla; or, Cadila Co.]. The corresponding daily dose of vitamin D3, which is significantly higher than that normally recommended in Germany (800 IU per day) and in other countries around the world such as the US (2000 IU per day), is commonly prescribed as a therapeutic dose in India possibly due to the popularity of vegetarianism. The reason for this choice of combined supplements above biological doses was as follows:

#### **2.1 Vitamin D**

Vitamin D3 (25-hydroxycholecalciferol) is the most bioavailable form of vitamin D for the human body and the bioactive form (1,25-dihydroxycholecalciferol) is synthesized by its enzymatic hydroxylation mainly in the kidney. This bioactive form of vitamin D also functions as a hormone that regulates calcium and phosphorus metabolism via a nuclear receptor that can alter the expression of genes in the intestine, kidney, and bone [29].

Vitamin D enhances cellular innate immunity partly through the induction of antimicrobial peptides, including human cathelicidin, and, defensins. Cathelicidins exhibit direct antimicrobial activities against a spectrum of microbes including many types of bacteria, enveloped and nonenveloped viruses, and fungi. The main action of these host-derived peptides is to kill the invading pathogens by perturbing their cell membranes. Moreover, it is effective in reducing concentrations of pro-inflammatory

cytokines that produce the inflammation that injures the lining of the lungs leading to pneumonia during viral infections like COVID-19 and increasing concentrations of anti-inflammatory cytokines [3].

Vitamin D deficiency is a worldwide problem, but is particularly pronounced in the elderly, who are at the greatest risk of contracting severe COVID-19 infection. The release of pro-inflammatory cytokines is one of the major causative factors in serious COVID-19 infections. However, vitamin D modulates its presence in the body by preventing macrophages from releasing too many inflammatory cytokines and chemokines. Calcitriol has also been found to exert an influence on ACE-2 receptors. Thus, it is not surprising that vitamin D deficiency has been correlated with COVID-19 cases and an increased risk of mortality in a European study [30]. Conversely, medical doctors in Eastern Europe have rarely found COVID-19 patients with vitamin D sufficiency to require ICU stays in hospital (personal communication from Dr. Martin von Rosen, MD).

#### **2.2 Zinc**

RNA synthesis occurs in the life cycle of the SARS-CoV-1 virus to reproduce its genetic material and is catalyzed by an RNA-dependent RNA polymerase, which is the core enzyme of a multiprotein replication/transcription complex. In the case of SARS-CoV-1, an excess of intracellular zinc ions has been found to efficiently inhibit the RNA-synthesizing activity of this replication and transcription multiprotein. Enzymatic studies in vitro have revealed that zinc directly blocks the activity of the RNA polymerase by inhibiting elongation and reducing template binding. This RNA polymerase core, which is a central component of the coronaviral replication/transcription machinery, is well conserved among the members of the coronavirus family including SARS-CoV-2 [31, 32]. Therefore, it is quite possible that zinc treatment would have a similar biochemical effect on SARS-CoV-2 and interfere with its ability to replicate.

In the human body, zinc displays antiviral effects by modulating the type I Interferon response and performs a variety of vital antioxidant functions [33]. Inside the cell, the harmful effects of free radicals are balanced by the action of antioxidant enzymes (such as copper-zinc superoxide dismutase) and non-enzymatic antioxidants (such as metallothioneins), which utilize zinc and help to regulate its intracellular levels [34, 35]. There are several other ways zinc functions in both adaptive and innate immune systems, as well. It regulates the proliferation, differentiation, maturation and functioning of lymphocytes, and other leukocytes. In addition, zinc regulates the immune response, and its deficiency increases susceptibility to inflammatory and infectious diseases, including pneumonia. Moreover, zinc deficiency may be present in 17% of the world's population [36]. Interestingly, a trial with four COVID-19 patients suggested that therapy with high dose zinc salt oral lozenges [up to 200 mg/day] initiated a significant reduction of disease symptoms within 24 hours [37]. Short-term zinc use at these doses is considered safe [38]. Thus, low-risk ways of increasing zinc bioavailability in the body above biological levels can be safely considered.

#### **2.3 Vitamin C**

Vitamin C is known as an essential anti-oxidant that efficiently quenches damaging free radicals produced during normal metabolic respiration by the body and it

*COVID-19 Prevention through Vitamin C, D, and Zinc Supplementation: A Small Clinical… DOI: http://dx.doi.org/10.5772/intechopen.103963*

functions as an enzymatic co-factor for physiological reactions such as hormone production, collagen synthesis and immune potentiation [39]. In addition, the antiinflammatory action of ascorbic acid is evidenced in several cytoprotective functions under physiological conditions, including the prevention of DNA mutation induced by oxidation. In fact, it has been established in *in vivo* studies that the consumption of vitamin C-rich foods is inversely correlated with the level of oxidative DNA damage [40]. Moreover, vitamin C is a well-known anti-viral agent that has been demonstrated to show anti-viral immune responses, especially against the influenza virus at the onset of infection by the increased production of IFN-α/β [39]. There has also been some interesting evidence that oral vitamin C (2–8 g/day) may reduce the incidence and duration of respiratory infections, while intravenous vitamin C has been shown to reduce mortality, ICU and hospital stays, and time on mechanical ventilation in severe respiratory infections. Vitamin C deficiency has been observed in many respiratory infections, as well, and a recent study from New Zealand reported that patients with pneumonia had depleted vitamin C levels as compared with healthy controls suggesting that it may be potentially helpful in the treatment of COVID-19 at therapeutic doses [41].

### **3. Results**

On average, the clinic was visited by 60 patients per day during July–December, 2020 and 45 patients per day during January–March 22, 2021. Thermal scanning was instituted at the clinic gates and any patient with a fever was seated outside and given a week's supply of vitamin C plus zinc tablets, vitamin D capsules, and aspirin. The patients without fever were allowed inside the clinic compound after receiving hand sanitizer and a disposable mask. They were instructed to keep a 2-meter distance between themselves and other patients as they waited on chairs outside the clinic. Only 6 patients were allowed into the clinic waiting room at one time (while 10–12 is the usual number). All patients with cold symptoms other than fever also received oral vitamin C/zinc and vitamin D3 supplements. All the hospital staff wore medical masks. PPE was not considered necessary as there were relatively few coronavirus cases locally and anyone with a higher than the normal temperature was not allowed inside the clinic. So, the hazard was not deemed to be extreme. Infected individuals in the district were immediately removed to pre-designated quarantine locations by government health inspectors where they received medical treatment for 2 weeks.

There were no adverse reactions to the special selective supplementation in any of the staff members during the first or second trial. There were no COVID-19 cases recorded among any of the staff members for the duration of this preliminary study, even though, approximately a third of them were living in and commuting from nearby towns and cities where the incidence rate was higher. The length of this trial suggests that there was ample opportunity for COVID-19 infection to occur in any of the subjects, especially since routine social distancing was not being observed during much of this time in India. All the subjects would have been exposed to potential virus carriers during work hours at the clinic (via asymptomatic carriers or those with cold symptoms other than fever), on public transport, and at home in their social circle. Similarly, the townspeople often traveled on public transport to other towns and cities and would have been exposed to potentially infected individuals at social gatherings.

### **4. Discussion**

Although, there is much interest in vitamin C, D, and zinc in the coronavirus literature, currently there is scant data about the potential synergistic role of these three supplements in COVID-19 prevention and clinical studies are lacking. Clinical trials for treating COVID-19 patients with these supplements are slightly more common; however, they usually do not focus on all three supplements together. Thus, the clinical study presented here appears to be the first of its kind [42].

A small two-part clinical trial with 14–15 subjects was undertaken to test the feasibility of taking specific supplements with anti-viral properties to aid in the prevention of COVID-19 infection, namely, vitamin C (500 mg/day), vitamin D (60,0000 IU/week), and zinc (20 mg/day) before the availability of any vaccine. It was concluded that this type of targeted supplementation of medical professionals and healthcare workers in an environment of potentially heightened exposure to coronavirus could be beneficial at the established dosages, which were non-biological doses well above corresponding biological doses. The combination of vitamins and mineral included in this preliminary study was selected for its special qualities in potentially combatting the coronavirus and was well-tolerated. On a biochemical level, the vitamin C likely acted by increasing the production of anti-viral Interferon α/β; vitamin D stimulated the secretion of antimicrobial peptides (defensins and cathelicidins) which perturb microbe cell membranes; and, zinc boosted the immune response in the subjects to ward off infection. Zinc may also have provided a secondary defense to clinic staff members by interfering with the SARS-CoV-2 replication machinery and disabling the viral RNA polymerase of invading virus particles. No other supplements aside from these three were provided to the participants to minimize confounding variables.

Even though, a variety of vaccines did become available following this study and all the clinic staff opted to be fully vaccinated (the vaccine supplied to healthcare workers in India was mainly Covishield), it was decided to continue with this special supplementation regimen (the vitamin D3 dose was gradually reduced to 30,000 IU per week), even after the six-month and two-and-three quarter month trial periods for several reasons. Firstly, it is not known, yet, exactly how long the antibody immunity generated by these vaccines lasts (although an estimate of 3–6 months has been suggested) and, therefore, there may be a lag period during which recipients are not protected. Lasting immunity following acute viral infection is variable and pathogenspecific ranging from life-long for smallpox or measles, to highly transient for common cold coronaviruses (CCC). It often requires maintenance of both serum antibody and antigen-specific memory B and T lymphocytes. Secondly, as new variant strains of the novel coronavirus continue to appear and change beyond recognition, many of the vaccines may become less effective or even ineffective at some point as antibodies can no longer recognize their corresponding antigens. For example, the Alpha, Beta, and Delta coronavirus mutant strains appear to have a modified spike protein with an increased binding capacity. The Gamma variant carries some of the same spike mutations [23]. This feature may render vaccines that target solely the novel coronavirus spike protein irrelevant. Evidence is already emerging that suggests fast-spreading coronavirus variants like Omicron with an ever-increasing number of multiple mutations in the spike protein may evade the main immune responses triggered by many vaccines and natural infection [43–47] possibly excluding T cell immunity [48, 49]. Thus, it is preferrable to seek protection simultaneously from several biochemical sources that disable different parts of the viral machinery. Thirdly, even though it is

#### *COVID-19 Prevention through Vitamin C, D, and Zinc Supplementation: A Small Clinical… DOI: http://dx.doi.org/10.5772/intechopen.103963*

possible to alter existing vaccines to target these new variants, it takes some time to re-engineer them, so it is not desirable for recipients to have an unprotected interval. Finally, there is the question of how many vaccines an individual can safely receive each year without engendering negative physiological consequences or increasing the chances of experiencing long-term side-effects.

As an example, *thimerosal* is a mercury-containing organic compound (approximately 50% mercury by weight) that has been widely used as a preservative in many inactivated-virus vaccines since the 1930s [50]. Mercury is toxic to both animals and humans and is associated with several adverse health effects including anemia, cardiovascular disease, developmental abnormalities, neurobehavioral disorders, kidney and liver damage, and brain cancer [51]. Although, it is claimed that thimerosal is safe in small doses, it is unlikely that experiments have been conducted on human subjects receiving two to three or more vaccines per year as seems to be required in the case of coronavirus. Not all the new coronavirus vaccines like the mRNA vaccines contain thimerosal, but there may be other ingredients with unintended future consequences, which have not been adequately tested. Naturally, these could have the greatest negative impact on younger members of the population. Therefore, the most prudent approach for government health agencies to adopt may be to continue to offer annual booster vaccines for those 60 years or over and to other vulnerable members of society.

Further issues with the various available vaccines have also come to light. For example, in rare instances, the AstraZeneca COVID-19 vaccine has been linked to blood clots, while the Pfizer and Moderna vaccines have been associated with severe allergic reactions in rare cases [52, 53]. It may be possible to mitigate some of these illeffects by adjusting the vaccine dose according to a recipient's weight (for example, a person who weighs 45 kg might receive a lesser dose than someone who weighs 90 kg or double their weight). Moreover, COVID-19 vaccination is associated with a lower risk of several, but not necessarily all, COVID-19 symptoms in those with breakthrough SARS-CoV-2 infection including long-COVID features, renal disease, mood, anxiety, and sleep disorders [54]. However, there were no breakthrough cases of COVID-19 between April and December 2021 in any of the 14 hospital staff members who had been vaccinated, but simultaneously continued with supplementation. No mild cold-like symptoms were observed in any of the subjects either. It is also worth noting that none of the staff received a booster vaccine during this period, which would only become available to healthcare workers and medical professionals in January 2022. Thus, this study successfully spanned the rise of Alpha to Delta variants (*pre-vaccine*), while breakthrough cases were averted during the peak and decline of Delta and the onset of Omicron in India (*post-vaccine*).

Unfortunately, the small sample size (14, 15) of the test group in these trials could not be analyzed statistically in relation to the much larger population in the town, which merely formed a general basis for comparison. So, this may be regarded as an uncontrolled study without matched controls. Ideally, another private clinic of similar size with equivalent numbers of medical and non-medical staff members, who were not receiving supplementation, could have participated as the control group. A second similar placebo group might also have been informative. However, this would not be possible in any future studies as vaccines have become widely available to healthcare workers all over the country. At the same time, it is likely that the other hygienic practices adopted at the clinic during the pandemic may have contributed to the positive result. Moreover, the annual incidence of coronavirus cases was relatively low in the town as compared with urban centres and must also

be taken into consideration. Nevertheless, it is more than likely that the clinic staff were exposed to persons infected with the coronavirus during the study (initially, the original strain, followed by the Alpha variant, at least, according to the region, and, then, later, the Delta variant), but local sequencing data was not available. Therefore, some interesting insights into supplementation with specific vitamins and minerals of medical personnel may have been gained as a result of these initial findings as no COVID-19 infections occurred among the *unvaccinated* hospital staff between July 1, 2020 and March 22, 2021 (**Tables 1** and **2**). No breakthrough infections occurred either in the staff members, who were *fully vaccinated* with the Covishield vaccine, between April and December 2021 with continued supplementation (**Table 3**). These results may prove useful for further clinical research into COVID-19 prevention, but, due to the small sample size, future studies should be conducted with much larger test groups, equally matched controls, placebo groups, and a complete statistical analysis. It may be particularly relevant for lower-income countries without immediate access to vaccines always and as an added precaution for healthcare professionals at higherthan-average risk of infection. This is also especially applicable in the event of waning vaccine efficacy as may be the case with the Omicron variant and some of its subvariants, which seem equipped to evade antibody immunity (not necessarily T cell immunity), cause breakthrough infections, and initiate reinfections [55].


#### **Table 1.**

*Oral supplementation of staff members—Part I; pre-vaccine (July 1–December 31, 2020).*


#### **Table 2.**

*Oral supplementation of staff members—Part II; pre-vaccine (January 1–March 22, 2021).*

*COVID-19 Prevention through Vitamin C, D, and Zinc Supplementation: A Small Clinical… DOI: http://dx.doi.org/10.5772/intechopen.103963*


#### **Table 3.**

*Oral supplementation of staff members—post-vaccine (April 1–December 31, 2021).*

Finally, there is no doubt that there will be ever new SARS-CoV-2 variants in the future, which may be less virulent, or possibly more so. As the virus evolves, these variants may become more transmissible and even better able to evade vaccine and natural immunity. These currently unknown mutants are beyond the scope of this book or chapter. However, the approach remains the same and we must be prepared with an artillery of weapons against the novel coronavirus rather than just relying on one.
