Cultured Cells, Dr. Fauci, and a Bold Hypothesis to Explain Why Hydroxychloroquine Alone Doesn't Work for COVID-19

How did the notion arise that hydroxychloroquine could be used against the SARS-CoV-2 virus that causes COVID-19? I want to spend some time on this because it involves an article that is often quoted to slander Dr. Fauci.
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Vero E6 Cells in Culture and Hydroxychloroquine

In 2005 there was a study where chloroquine (a drug related to hydroxychloroquine) was found to be effective at reducing infection with the virus, SARS-CoV (which is 79% related to the COVID-19 virus, SARS-Cov-2), in primate cells in culture. Early on in the COVID-19 pandemic, it was claimed that Dr. Fauci was lying when he said that there was no good evidence for the effectiveness of hydroxychloroquine against COVID-19 because he has known about this article for 15 years. This claim is still being made over and over in social media, and has even been retweeted by some high profile people in the hydroxychloroquine debate.

Any scientist who has worked with cells in culture (for example, me) will tell you that extrapolating from cultured cells to the full organism is fraught with peril. The metabolism of cells in culture and the way they react to chemicals or biologicals can be substantially different from the way they react as part of the full organism. In the full organism, cells are in a three dimensional matrix, interacting with other cells types and exposed to the daily cycles of fluctuations in levels of nutrients, hormones, temperature, oxygen, etc. Also, any drugs delivered to the organism, depending on the mode of delivery, may have to pass through several layers of other cells before they reach their intended target. In culture, the cells are in a two dimensional environment interacting only with cells like themselves under fixed conditions. When you deliver a drug to the cells in culture, it reaches them right away. So, more often than not, what you get using cell culture, is different from what you get in the full organism.

Additionally, the cells used in the study mentioned above were Vero E6 cells. This is a cell line derived from the kidney of an African Green Monkey, and these cells even have a different number of chromosomes compared to those of the original monkey. Infecting cultured cells from the kidney (not the airways) of a monkey (that are different from human cells and even from the cells of the original monkey) with the SARS-Cov virus (which is different from the COVID-19 virus), treating them with chloroquine (which is different from hydroxychloroquine), and then extrapolating the results to the full human being to argue that Dr. Fauci “…has known for 15 years that chloroquine and it’s even milder derivative hydroxychloroquine (HCQ) will not only treat a current case of coronavirus (“therapeutic”) but prevent future cases (“prophylactic”).” is scientifically inaccurate, to say the least.
 
Then why do scientists use these monkey cells to test compounds against viruses that infect humans? The answer is that these cells can be infected with many viruses and they are a convenient, but very preliminary, test that can be used to screen many compounds quickly, and identify those that display activity against viruses. Once a compound is identified, it is subjected to more complex tests that may involve human cells and studies with whole animals to see if it still works. As it turns out, hydroxychloroquine has been recently been tested with these Vero E6 cells, and it has been found to have antiviral activity against the SARS-CoV-2 virus. But can hydroxychloroquine stop the COVID-19 virus from infecting cells from the human airway, which is the main site of entry of the virus into the body?
 
A study performed with reconstituted airway epithelium developed from primary human nasal or bronchial cells indicated that hydroxychloroquine had no activity against the COVID-19 virus. Why would this be the case? A group of researchers have proposssed a very clever answer to this question.
 
There are two ways that the COVID-19 virus can get into a cell. One involves the uptake of the virus into an intracellular compartment called the “endosome”. The endosome is a compartment that is acidic which is optimal for the functioning of an enzyme called Cathepsin L (CatL) that acts on the virus and activates it. One of the effects of hydroxychloroquine is to lower the acidity of the endosome which impairs the activity of CatL and thus hinders the activation of the virus. However, the cells of the human airway epithelium have low levels of CatL. In these cells, the entrance of the virus is achieved through a second route involving activation by an enzyme called TMPRSS2 which is not dependent of endosomes or acidity and is therefore not affected by hydroxychloroquine. Thus the CatL (hydroxychloroquine sensitive) pathway is predominant in Vero E6 cells, whereas the TMPRSS2 (hydroxychloroquine insensitive) pathway is predominant in the human lung cells. If you use Vero E6 cells that have been genetically engineered to express the TMPRSS2 enzyme (and thus have the TMPRSS2 pathway too), then hydroxychloroquine loses its ability to inhibit the COVID-19 virus infection of the cells! Other researchers have also found that the inhibition of SARS-CoV-2 entry into cells by hydroxychloroquine is antagonized by the presence of the TMPRSS2 pathway.
 
The above is a bold hypothesis that explains why hydroxychloroquine alone has not been found to work in the best-designed studies (1, 2, 3, 4, 5, 6, 7) performed so far. The reasoning is that it does not work because it doesn’t affect the TMPRSS2 pathway of COVID-19 virus activation that is the predominant viral activation pathway in human airway cells! This hypothesis also eliminates the original rationale to use hydroxychloroquine, which arose from using Vero E6 cells as a screen, and it shows how wise Dr. Fauci was in not giving a lot of importance to that very preliminary 2005 study.
 
Nevertheless, notice I mentioned that the above proposal is just a hypothesis. Some people have interpreted the results to mean that hydroxychloroquine reduces the COVID-19 viral infection in monkeys, not humans. However, hydroxychloroquine does not reduce or prevent COVID-19 infection in macaque monkeys. More studies are needed before we can conclude that this is the reason why hydroxychloroquine doesn’t work in humans.
 
However, if you are one of the people that argue that hydroxychloroquine by itself is not active (has no antiviral activity), but rather that it enhances the uptake of zinc which has the real antiviral action, then this hypothesis does not affect your claim. Whether zinc acts with hydroxychloroquine to ameliorate or prevent COVID-19 remains to be demonstrated in well-designed clinical trials (and several are ongoing). But what we can say right now is that the best evidence we have so far indicates that hydroxychloroquine IS NOT effective by itself or with antibiotics against COVID-19, and it’s time we started accepting this.
 
Micrograph of Vero Cells under green light (100X) by Y tambe is used here under an Attribution-Share Alike 3.0 Unported license. This micrograph was merged with an image of hydroxychloroquine by Fvasconcellos which is the public domain.

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