The effects of orally administered lactoferrin in the prevention and management of viral infections: A systematic review

Summary It has been demonstrated that lactoferrin (LF) plays a role in host defence, but evidence on its potential antiviral property from clinical studies is fragmented. Our systematic review aimed at identifying the effects of orally administered LF against virus infections. The systematic search was conducted on PubMed, Scopus, Web of Science, BioRxiv.org and ClinicalTrials.gov from database inception to 7th January 2021. Eligible articles investigated any virus family and provided data on the effects of orally administered LF of any origin in the prevention and/or management of confirmed viral infections in people of any age. A narrative synthesis of the results was performed. Quality was assessed with the Cochrane Risk‐Of‐Bias and ROBINS‐1 tools. A total of 27 records were included, nine of which were registered protocols. We found data on Flaviviridae (n = 10), Retroviridae (n = 3), Coronaviridae (n = 2), Reoviridae (n = 2) and Caliciviridae (n = 1). Most published trials were at high risk of bias. The findings were heterogeneous across and within viral families regarding virological, immunological and biological response, with no clear conclusion. Some weak but positive results were reported about decrease of symptom severity and duration, or reduction in viral loads. Despite high tolerability, the effects of LF as oral supplement are still inconsistent, both in preventing and managing viral infections. Small sample sizes, variety in recruitment and treatment protocols, and low study quality may have contributed to such heterogeneity. Better‐designed studies are needed to further investigate its potential benefits against viral infections, including SARS‐CoV‐2.


| INTRODUCTION
Lactoferrin (LF) is a multifunctional glycoprotein, member of the transferrin family, 1 identified for the first time in 1939 in bovine milk and isolated in 1960 from human milk. 2,3 Several studies have demonstrated that it plays a role in host defence 4,5 ; because of its structure, it is a component of the innate immune response and a potent immunomodulator. 6 Its ability to bind free iron ions 7 prevents the tissues from excessive inflammatory processes. 8 Additionally, it is now widely recognized for antioxidant activity 9,10 and antibacterial activity, 11 and Bezault et al. presented convincing data about its anticancer activity in murine models of fibrosarcoma and melanoma. 12 In the 1980s, some authors documented for the first time that LF may also affect the myelopoiesis of mice inoculated with a friend virus complex, 13 paving the way for other hypotheses about the role of LF in viral infections. 14 To date, the antiviral property of LF has been confirmed by several in vitro studies. [15][16][17] It is directed against a broad spectrum of viruses, including both RNA-and DNA-viruses, enveloped as well as naked viruses. 15 Some studies have indicated that LF prevents infection of the host cell, rather than inhibiting virus replication in the target cell, 18,19 whereas other authors have demonstrated its ability to prevent viral infections by acting through interaction with heparan sulfate proteoglycans 20 in a dose-dependent effect, 21 by binding to viral particles or viral receptors, and by involving apoptosis or inflammatory pathways. 15 Another mechanism of action is the upregulation of the antiviral response of the immune system. 22,23 In fact, NK-cells, monocyte/macrophages and granulocytes play an essential role during the early phases of a viral infection, 24 and polymorphonuclear leukocytes seem to become more effective after exposure to LF, thanks to greater motility and faster production of superoxide. 25,26 Over the last year, the severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) has captured the attention of the scientific community. With the rapidly escalating situation worldwide, researchers have sought treatment strategies to control this infection. 26,27 As vitamin and mineral insufficiency was observed in COVID-19 patients at increased risk of mortality, 28 dietary supplements and drugs have been considered as supportive therapy. 29 Relying on some positive results, 23,30 some authors proposed LF as a supplemental treatment for COVID-19, 31 but evidence on its potential effects from clinical studies is still fragmented. The aim of our systematic review was to identify the effects of orally administered LF against viral infections, with a specific focus on those caused by coronaviruses, to provide a synthesis of the results and support clinicians in the evaluation of supplemental treatments for COVID-19. Additionally, BioRxiv.org was searched as a pre-print database using the string 'lactoferrin AND virus' and 'lactoferrin AND SARS-CoV-2' whereas ClinicalTrials.gov was queried to map planned, ongoing or just completed clinical studies. The same screening process mentioned for the bibliographic databases was applied.

| Data collection and synthesis
For each eligible study retrieved from the literature search (i.e., published or pre-print), two reviewers independently extracted the following information: first author, year of publication/submission, country, virus family, characteristics of the target population, study design, type and duration of the intervention, area of evaluation (prevention or management of viral infections), main findings and side effects. Articles were grouped according to the virus family, and a narrative synthesis was performed. As for the records investigating the LF effect on the management of viral infections, three categories were considered: virological response, immunological response and biological response. Two independent authors performed the quality assessment of the articles included in the systematic review using the revised Cochrane Risk-Of-Bias tool version 2 34 for randomized studies and the ROBINS-I tool 35 for non-randomized interventions.
Discrepancies were resolved by consensus or by a third reviewer.
Judgements on the quality of the studies followed the Cochrane guidelines. 34,35 For each record retrieved from ClinicalTrials.gov, two reviewers independently extracted the following information: principal investigator and identifier, title, country, start and completion date, condition or virus being studied, purpose/outcome, recruitment status. A narrative synthesis of the results was performed.

| RESULTS
After the removal of duplicates, 2822 records resulted from the initial search ( Figure 1). Screening by title and abstract selected 162 articles eligible for full-text analysis, from which 148 records were excluded with reasons. Four records were added to the previous 14 from the reference lists of relevant articles, and nine records retrieved from ClinicalTrials.gov met our inclusion criteria, for a total of 27 records included in the systematic review.

| Flaviviridae
Three studies compared a different daily dosage of bLF, 38,46,47 three studies compared bLF to no therapy, 37,39,48 two studies compared a combination of bLF and IFN-α or a combination of bLF, IFN-α and ribavirin to the same therapeutic regimen without bLF, 40,41 one study compared bLF directly to ribavirin plus IFN-α 45 and one study both bLF versus placebo as well as bLF-ribavirin-IFN-α triple therapy and ribavirin-IFN-α therapy. 36 The bLF dose varied consistently across the studies, from a minimum of 0.4 g/day 38 to a maximum of 7.2 g/ day 46 (Table 2).
All studies analysed the virological response after treatment in terms of HCV-RNA, with heterogeneous results: one study 38 found a significant decrease in the viral load after a higher bLF dosage, whereas one study 46 reported a non-significant dose-response effect. Tanaka and colleagues 47 described a reduction in the outcome only for patients with low pre-treatment viral load. Most studies reported a non-significant difference in the virological response between bLF and placebo, 37,39,48 as well as comparing the additional effect of bLF on a drug therapy 40,41 or considering bLF in direct comparison to ribavirin plus IFN-α. 45 Only one study reported a significant decrease in the viral load after bLF in monotherapy or in triple therapy, but among patients classified as bLFresponders. 36 The immunological response was investigated in three studies, 37,39,45 with no consistent results on IL-18 levels. 37,39 Oral administration of bLF did not seem to influence IL-4 and IFN-γ levels, 37 whereas El-Ansary and colleagues 45   Eight studies reported the effects on biological response, mainly in terms of ALT levels. No change was observed according to the bLF dosage, 38,46 or in general by most studies. 37,[39][40][41] Only one study reported a significant decrease in ALT level 48 and plasma 8isoprostane levels compared to no therapy, while one study found a decrease in ALT serum concentration only in patients with low pretreatment viral load. 47 All studies but two 45,48 provided data on side effects. Three studies reported none, 37,38,47 in one study the signs and symptoms reported were minor but dose-dependent, 46 whereas in the last four studies the authors did not find any difference between the side effects shown by the patient groups. 36,39-41

| Caliciviridae
The only study on calicivirus compared the oral administration of bLF at a dosage of 0.5 g/day to placebo 44 ( Table 3). The potential role of bLF in preventing episodes of diarrhoea in children was the main outcome, but no reduction in diarrhoea incidence was reported.
Rather, a decrease in duration and severity of gastroenteritis-related symptoms was observed.

| Coronaviridae
One of the two studies that enrolled patients with COVID-19 is still in the pre-print version 52 (Table 3)  found a significant decrease in the viral load during bLF administration in patients that received no antiretroviral therapy (group I) or considering those who received a combination of two antiretroviral agents (Group II), with no difference between the two groups. The immunological response was evaluated in all three studies but heterogeneously. A differentiation of subpopulation T-lymphocytes and an improvement in phagocytosis and killing, Toll-like receptor expression, and IL-12/IL-10 ratio were found in one study 53 ; CD8+ cell count was assessed once, with no meaningful findings 49 ; absolute CD4+ cell count seemed to not improve in two studies, 49,53 whereas one study 49 found an increase in the CD4+ cell percentage in groups I and II during bLF administration, even though it was higher for the latter. The study that compared rh-LF and the placebo group did not highlight a significant difference in immunological response. 42 Symptoms related to the underlying disease were evaluated in two studies 42,53 without significant results. The most recent paper also studied intestinal microbiological effects, reporting no significant differences between the two groups but an increase in transferrin saturation. 42 Lastly, side effects were found to not differ between the two groups in one study. 42

| Reoviridae
Reoviridae were analysed in two studies that compared the effects of bLF administration at a daily dosage of 70-80 mg 43 and 100 mg 50 versus placebo (Table 3). No significant differences were found between the two groups in preventing gastroenteritis onset in both trials, whereas they yielded heterogeneous results for the assessment of the symptoms. Also, the immunological response was investigated in one study only, 43 with no clinically meaningful findings.

| ClinicalTrials.gov
The nine records retrieved from ClinicalTrials.gov were randomized trial protocols (Table 4). Three were registered in Egypt, two in the The remaining four protocols focus on SARS-CoV-2 but are reported as not yet recruiting.

| DISCUSSION
The rapid spread of the SARS-CoV-2 virus has brought the scientific community to consider all potential therapeutic agents 54 and evaluate or re-evaluate every possible support therapy. 55  In most studies, the glycoprotein was tested in relation to the management of infectious diseases only. This was not unexpected since the eligibility criteria of our systematic review required confirmation of the virus, more easily obtained in chronic conditions.

T A B L E 3 Main effects of lactoferrin oral administration in the prevention and management of viral infections
Author Allen, NCT01092039 XIGO effectiveness study: An investigation of the safety and efficacy of oral XIGO tablets on patients diagnosed with the common cold Completed and published 42 Campione, NCT04475120 Interventional pilot study to assess the use of oral and intra-nasal liposomal LF in COVID- 19  However, a consistent heterogeneity in the findings was observed, both among viral families and within the same family. Flaviviridae was the most frequently investigated, and all studies focused on HCV, the major cause of liver disease worldwide, that leads to chronic carriage in 70%-80% of cases with the risk of development of complications such as cirrhosis and cancer. 58   Lastly, as confirmation of a viral infection was an inclusion criterion, it is possible that we may not have included a few data on the effects of LF on the infections in which the etiological agent was not specified.
However, it was impossible to be sure about the infectious source given the low specificity of the symptoms, and our focus was limited to the glycoprotein's antiviral activity.
To conclude, in vitro studies show evidence in favour of a protective role of LF. However, despite its relatively safe profile, the results from clinical trials investigating LF oral supplementation are still inconsistent, both in preventing and managing these infections. In our opinion, this could be the result of a combination of factors including, but not limited to, small sample sizes, heterogeneity in recruitment and treatment protocols, and low study quality. Hence, further research is needed to better investigate the potential benefits of LF oral administration in relation to viral infections, including SARS-CoV-2.

ACKNOWLEDGEMENT
The authors gratefully acknowledge Paul Griffiths, Professor of Virology, University College London, for reviewing, suggestions and editing.
Open Access Funding provided by Universita degli Studi di Roma La Sapienza within the CRUI-CARE Agreement.