Carmen Mirabelli, Ph.D, a virologist, was part of an immunology team at the University of Michigan in the States that released a ground-breaking study in 2021. At a time (pre-vaccine) when the world was looking for solutions to a devastating global pandemic, the team screened 1,425 FDA approved drugs and compounds in an attempt to find one that might be effective at blocking SARS-CoV-2 in vitro. These included Hydroxycholorquine – an anti-malarial drug that has been used in autoimmune conditions such as rheumatoid arthritis and lupus – and Remdesivir, an anti-viral drug that prevents virus multiplication. The team at Michigan threw Lactoferrin into the mix and were amazed when it beat every other contender. Carmen presented the results of her study at a 4-day Lactoferrin conference at the end of last year and had Lactoferrin experts and scientists on their edge of their seats. She spoke to Leapfrog’s founder, Stephanie Drax, about the team’s findings and the therapeutic potential of this wonder molecule.

Can you tell us why the University of Michigan conducted this study and what you were hoping to achieve?

I’ve been studying viruses for the last 10 years – particularly viruses of the intestine – and also have a special interest in cellular response to infection and antiviral development. When Covid hit all our efforts in the virology community were directed to finding something that could combat this viral infection that had dramatically changed our lives. So, we put together a task force at the University very quickly in March 2020, right after SARS-CoV-2 was declared a pandemic, and I started to work with group of amazing people. We developed an infrastructure from the bottom up to study this virus. In my career, I’ve never seen such a concerted effort of scientists pitching in to help. Funding came from various sources including the Chan Zuckerberg Foundation and internal funding from Michigan that came from the National Institute of Health, so even economically it was a concerted effort – the screening platform that we used was very expensive so it was great to get all this support. I was asked to help because not only am I a virologist – with knowledge especially of viruses that are very dangerous that are manipulated in the lab – but also because I’ve done a lot of antiviral development. So, I had the skill set that the university was looking for. I was responsible for the set up of the assay – the experiment – and for performing the screening of the compounds and drugs. It was a procedure that’s called ‘repurposing’ that is done regularly in drug development; to develop a drug takes over 10 years, and in a pandemic setting you don’t have that time. The thinking of the University was to take drugs that already exist – and that we know are not toxic – and see if by any chance they retain some anti-viral activity against SARS.

Why did you include Lactoferrin in the line-up?

Our selection was not only FDA-approved drugs but also compounds that we thought could be interesting. We put Lactoferrin in the mix because we had some previous experience with Lactoferrin having anti-viral activity against our favourite virus – the intestinal virus, or norovirus – with data that had been published one year before the pandemic. Also, we knew that Lactoferrin was active in vitro against SARS-CoV-1 from 2003, and also it has very interesting antimicrobial activities.

Tell us how the study worked?

The experiment studied viruses via their ability to kill cells. The method that we used is called Immune Florescence Imaging – we grow cells, put the virus on cells and quantify the infection. We used a virus that was florescent, to show the virus growing or multiplying – showing the florescence in the cells and neighbouring cells. We took cells from human origin: one line that came from the liver and one line that came from the lung (which is relevant as these are cells that SARS targets). We put the cells on plates and added a select amount of virus for us to know how much we would have in 24 hours and 48 hours. It was very controlled, so that the more the virus multiplied, we could actually see the cells that were affected and count them. We expected that 48 hours post infection we would have 30% of infected cells, and we were looking for the compound or drug that would reduce the infection or bring down it right down to 0%. If we added drugs and then had less virus than we expected 48 hours later for instance, then we could say that this drug is active against the infection.

What did you discover?

We used Lactoferrin as a pilot experiment to set the conditions and we were amazed because the moment we put Lactoferrin there, we didn’t see any virus anymore – we couldn’t detect any florescence. And we repeated and repeated it many times – and on different cells and different Covid strains such as Alpha, Beta, Delta variants – and it was active across the board. We were so excited by our findings, we wanted to publish the results in that first week, before we’d even started on the other compounds and drugs. And we also tried different types of Lactoferrin; it’s a protein that binds iron, so we tried Lactoferrin without iron and with iron, and recombinant Lactoferrin and bovine Lactoferrin, and it worked every time. So, there is something that it does that is regardless of its origin. We could see that it was really active even compared to Remdesivir – a drug that had been approved against Covid – and it was more active than Hydroxychloroquine, which was not active in our assay or in vivo with patients. So, we tried to mix it with Remdesivir and they had a nice synergistic effect. We were so impressed, we started taking Lactoferrin ourselves and giving it to our families.

How does Lactoferrin have this extraordinary effect?

We narrowed down the effects on virus replication by studying more deeply and found out that Lactoferrin could compete with the virus for the binding of the cell receptor. The virus binds on the cell first and then is internalised for the replication to start. Lactoferrin blocks at the cell’s binding site, and for a drug to block at this level is very important because it means that the virus won’t even get into the cell. The other cool thing is that even when we added Lactoferrin after the virus had entered the cell, we could still see a reduction of infected cells. So, it shows that Lactoferrin really has different modes of action – not only does it block the entry, but also once the virus is in, it blocks replication. We think that it blocks replication by up-regulating, or increasing, the expression of the innate immune response of the cells, some factors that, like sentinels, the cells produce when they encounter a foreign infection. This was very interesting, and we found these different modes of action by performing different experiments. We confirmed this effect of Lactoferrin increasing the sentinels by putting Lactoferrin on healthy cells with no virus present to see how the cells reacted to Lactoferrin, and they started to produce these sentinels a lot.

Our results showed that Lactoferrin can really prime cells, so that they are more ready to fight if something comes into the body. And this is a super interesting property of Lactoferrin.

We talk a lot about immunomodulation, which is the ability of some molecules to charge up the immune system when its needed – like in the innate immune system signals – but then calm it down when it becomes systemic. The immune system isn’t something that’s stable – it’s something that goes up when its stimulated, but then it has to calm down. The severe consequence of SARS at the systemic level is due to an exuberant immune systemic response that somehow is not controlled anymore by our body. And the cool thing about Lactoferrin is that it has this capability systemically to bring down the level of cytokines.

Lactoferrin can help the infection in both ways: locally, when the cells see the virus and bringing down replication, but also systemically, trying to bring the body back into equilibrium. Think of a wound – you want it to be inflamed, but then you want it to heal.

What trials would you like to see in vivo to back up your results in vitro?

We love Lactoferrin, but we do need in vivo studies (in humans) to back up these in vitro findings. We don’t know yet if Lactoferrin is concentrated enough to block infection in vivo. We didn’t need much Lactoferrin to have an anti-viral effect – in vitro we needed 50 times less Lactoferrin than is present in human milk – but we’re not sure that Lactoferrin will be at the site of infection at this level of concentration in vivo.

It would be nice to have a cohort of people who’ve been using Lactoferrin for years so that we could see after 10 years what the incidence of infectious disease was in these people, versus people who have not taken Lactoferrin ever. Or we could do a small clinical trial in the case of infectious disease, or Covid – for example a household trial with someone who has Covid or the flu, and then we put other people in the house on Lactoferrin. The University of Michigan wanted to conduct some trials in schools, care homes or with doctors and nurses on the front line who were most exposed to Covid. The limitation though is to produce a control arm of placebo, non-treated individuals. It has to be well planned to draw conclusions and see if there are differences in the outcome.

We have a chewable tablet for Leapfrog IMMUNE and we understand that the University of Michigan is considering a chewable tablet format for their own trials?

Yes, the university wanted to create a chewable tablet because the pharmacology department has experience with enteroprotected capsules and believed that the body would absorb a chewable tablet better than having the capsules that could be broken in the very low pH of the stomach. Then the protein may not be released systemically where it should go.

What benefit could Lactoferrin offer as an anti-viral today?

It’s very important, with SARS in particular, that we invest more time and money into the development of small molecules. It’s important in the control of local epidemics moving forward, or in immune compromised patients for whom the vaccine doesn’t work, or in the case of small kids who can’t be vaccinated yet. So, it’s good to have an anti-viral together with a vaccine because the advantage of anti-virals is that they will block infection so you won’t even experience symptoms and you will block transmission immediately. We need to get people excited and get money to move forward. Lactoferrin is a story that’s known in the scientific world and we need to push it further – leveraging this great effect against SARS and other infections – especially as it has no side effects and there is a lot of potential.