Beyond Vaccines

In a year dominated by an RNA virus largely unknown 12 months ago, it is perhaps not surprising that the 2020 Nobel Prize for Medicine was awarded to three researchers involved in the identification of the blood-borne pathogen that sickens and kills people globally on a scale only seen with HIV and tuberculosis: hepatitis C virus (HCV).

From Harvey Alter’s definition of a non-A, non-B chronic hepatitis agent to Michael Houghton’s first isolation of viral nucleic acid fragments to Charles Rice’s final proof that the isolated HCV was the infectious agent, the identification of the virus allowed researchers and clinicians to develop diagnostic tests and drug cocktails to reduce infection rates around the world.

Standing on the shoulders of those scientists, researchers and clinicians today have had to accomplish in less than a year the same feats that took Alter, Houghton, Rice and their many colleagues 25 years.

And the world is on the cusp of several vaccine candidates showing more than 90-percent effectiveness against SARS-CoV-2.

But even as everyone waits for that hallowed day, more than 250,000 people have died of COVID-19 in the United States. Almost 170,000 in Brazil. 132,000 in India. And 100,000 in Mexico.

1.34 million people have died around the world because vaccines are not yet available and effective treatment is largely lacking.

Vaccine variables

“The conversation has all been about vaccines because really, people just want this to go away,” says Vikram Sheel Kumar, CEO of Clear Creek Bio. “The problem is that COVID-19 is not going away soon, so we need medicines for if and when we do get infected.”

Terina Martinez, field applications specialist at Taconic Biosciences, breaks the challenges down even further.

“We have eight billion people on the planet,” she says. “Operationalizing and implementing a vaccine at that level is going to take actually a couple of years probably. So, even on the timeframe of the sheer effort that it will take to vaccinate a global population, we will still have people who get infected and we will still need to provide improved therapeutic approaches.”

And even when vaccines roll out, she presses, low adoption rates continue to be a problem in the United States as seen annually with influenza vaccine. And in cases where immunization requires two doses, there will be a time frame when people are still vulnerable.

And Kumar raises the further question of how long it will take to develop immunity once vaccinated or how long the immunity will last, questions that are only now being answered in clinical trials.

“For all of those reasons collectively, I think that much attention is warranted in the preclinical space to develop targeted therapies,” Martinez concludes.

“One of the ways I like to think about this is that vaccines, in some respects, are offense,” Kumar stresses. “We absolutely need them, and I think it's right that we're investing time and money in them. But we know in sports and in life, we need to play defense, too. And that's really where I think antivirals come in. Ultimately, a safe and effective antiviral is what we're going to need, along with vaccines, to finally take off our masks.”

Targeting the virus

The most obvious target for antivirals development is the virus itself. Not only could they potentially prevent infection, but in avoiding the host cells, they could offer a better safety margin than host-targeting drugs.
A major challenge of targeting the virus, however, is the risk that a mutation might render the therapeutic completely useless, as is so often seen with antibiotics and cancer drugs.

To minimize the risk of a debilitating mutation, Cocrystal Pharma utilizes structural biology and X-ray crystallography. The company has focused its attention on structures within viral enzymes that are highly conserved among viruses within a family, paying particular attention to the viral replication enzymes.

 “We’re identifying those regions that are essential for the enzyme to function,” explains CEO Gary Wilcox. “And then using X-ray crystallography, we can look at the binding site of an initial inhibitor and have a near atomic resolution map of the binding interaction. We can use this map to design improved inhibitors that will become our drug candidates.”

Another important step in this process, however, is mutating the identified region of the enzyme to determine if viral replication will not occur.

“We’ve really set up a wonderful situation, because if a mutation occurs at that site, then the enzyme won't be active and therefore the virus won’t be able to replicate,” Wilcox enthuses. “And if there's not a mutation at that site, then our compound will bind to the site and the virus won’t be able to replicate. So, we’ve got a solution to drug resistance either way.”

From a safety perspective, it is also important that the target viral protein doesn’t have a human counterpart. By selecting a viral target that is unlike any human proteins, there is less likelihood for any associated toxicity reducing potential side effects of a drug.

In its coronavirus program, Cocrystal is targeting viral replication in two ways. First, it is developing inhibitors for the RNA-dependent RNA polymerase that the virus itself encodes to replicate its genome. In addition, Cocrystal is also targeting the protease that converts this polymerase from a pro-enzyme to its functional form.

This latter work arose from researchers at Kansas State University and their team developing treatments for coronavirus-infected cats that developed fatal feline infectious peritonitis (FIP).

For more than a year, Wilcox says, Cocrystal President Sam Lee had been interacting with the KSU team because of the possibilities their work has for coronavirus therapeutics.

In August, KSU’s Kyeong-Ok Chang and collaborators described their effort to expand the library of inhibitors of 3C-like protease (3CLpro) beyond the compound they had developed for FIP. Of the dipeptidyl compounds they synthesized, several showed potent activity against the SARS-CoV-2 3CLpro in both FRET enzyme assays and cell-based assays.

They then tested the compounds in mice infected with a mouse-adapted MERS-CoV strain (see “Clique of a mouse” after the end of this main article). They found that one compound reduced both lung viral load and lung pathology, although the degree of recovery depended significantly on how quickly mice were treated after infection.

“The MERS-CoV mouse model used here provides proof of principle regarding the therapeutic potential of our protease inhibitors for treating severe human respiratory coronavirus disease,” the authors reflected. “Limitations of the current study include differences in host receptor usage, mortality, and transmissibility between MERS-CoV and SARS-CoV-2.”

“Thus, further evaluation of our protease inhibitors in mice, hamsters, or nonhuman primates experimentally infected with SARS-CoV-2 will be crucial to assess these inhibitors as potential therapeutic options for COVID-19,” they continued.

In February, Cocrystal signed a licensing agreement with the Kansas State Research Foundation for rights to antiviral compounds KSU scientists had developed against both coronaviruses and noroviruses. In April, they extended that agreement to include more preclinical lead candidates.

“We are encouraged that administration of this compound significantly increased survival and reduced lung virus titer in the infected animals even when given one day after virus infection,” said Lee in the April announcement. “We are working tirelessly to leverage our proprietary drug discovery platform to advance this antiviral program and believe this new set of compounds has the potential to provide the lead compound for the program.”

Wilcox suggests that the dual approach used by Cocrystal to inhibit viral replication further reduces the risk of mutations leading to viral spread.

Beyond SARS-CoV-2, Cocrystal also has two programs against influenza, a norovirus and a hepatitis C program, all targeting polymerase complexes.

CC-42344 binds the highly conserved PB2 domain of the viral polymerase complex and has shown efficacy against influenza A, including avian pandemic and Tamiflu-resistant strains. The company is also working with Merck to develop another class of polymerase inhibitors against both influenza A and B.

In Phase 2 clinical trials described in 2019, Cocrystal’s non-nucleoside inhibitor (NNI) CC-31244 targeting hepatitis C virus NS5B polymerase showed efficacy against HCV infection when combined with Epclusa.

Trying to add to the viral protease inhibitor armamentarium, Timothy Spicer and colleagues at Scripps Research Institute and Calibr developed a high-throughput screen of repositories of clinic-stage or FDA-approved compounds.

Transiently transfecting cells with PLpro and a luciferase-based reporter, the researchers screened more than 150,000 compounds for molecules that would decrease luminescence. They then tested the compounds that made the first cut in enzymatic and cell-based assays to characterize their impact on PLpro as well as for signs of cytotoxicity.

The results were mixed.

“While we were successful at rapidly screening large libraries of drugs that may be repurposed, this target has proven to be refractory to identifying any bona-fide leads,” the authors noted. “This was somewhat surprising considering the robustness of the cell- and biochemical-based approaches and the considerable reproducibility of the hits from the vast diversity of drugs found in each library.”

“Ultimately, the cytotoxicity of the drugs against the same cells under the same conditions proved to be an excellent counter-screen to remove liabilities early on,” they added, taking solace in their failure to identify a lead. “It clearly limited the pool of drugs for further follow-up, however.”

As a self-described socially conscious biotech, Appili Therapeutics initially licensed its antiviral favipiravir (Avigan) from Fujifilm Toyama Chemicals to tackle lassa fever, an infection endemic to sub-Saharan Africa, explains CEO Armand Balboni.

“And then, of course, COVID-19 happened,” he recounts.

Already approved for treatment of pandemic influenza in Japan, and having shown some efficacy against Ebola, MERS-CoV and SARS-CoV, favipiravir was an obvious candidate for SARS-CoV-2. Favipiravir also inhibits viral RNA-depended RNA polymerase.

In March, Lei Liu and colleagues at The Third People’s Hospital of Shenzhen and Beijing Institute of Pharmacology and Toxicology compared the effects of favipiravir versus lopinavir or ritonavir in patients with COVID-19.

They found that not only did favipiravir induce viral clearance more quickly, but also it offered significantly better chest CT results.

“This finding suggests that improvement of the disease may depend on inhibition of the SARS-CoV-2, and that [favipiravir] controls the disease progression of COVID-19 by inhibiting the SARS-CoV-2,” the authors argued. “Since the infection of SARS-CoV-2 was thought to be self-limited and characterized by systemic inflammation reaction, symptomatic and supportive treatment was mainly recommended by the WHO and the National Health Commission of the People’s Republic of China.”

Balboni had already had some experience with favipiravir, exploring its use against Ebola as part of a U.S. Department of Defence fellowship.

“I also looked at remdesivir,” he says. “And I did do a little bit of work on the Merck program.”

He quickly came to realize that although there were a lot of studies underway, they weren’t the right studies. They were all too small, he says, to definitively answer whether the drugs would work against the virus of interest.

“So, I was very insistent from the beginning when I was speaking with Fujifilm that we would get to the deal terms when we got to the deal terms, but speed was really important here,” Balboni recalls. “But we had to be fast and good, and so we designed the trials as large randomized controlled trials—Phase 2 and Phase 3—to definitively answer whether or not Avigan was going to work against COVID-19.”

The company started with the highest risk population, subjects 50 to 65 years with comorbidities and those in long-term care facilities.

“Fujifilm donated some drug product to us at a time when it was very hard to get, and we went about designing and running trials at-risk,” he recalls.

In October, the company announced the dosing of the first patients in a Phase 2 study of favipiravir for COVID-19 outbreak control in long-term care homes in Canada. The goal here is to test the treatment as prophylaxis against infection. Thus, once infection is PCR-confirmed in a resident, all consenting residents in that unit will receive either favipiravir or placebo.

The primary outcome of the study will be infection control, defined as no new confirmed cases of COVID-19 for at least 24 days.
The company is also running several Phase 3 trials, looking at favipiravir in an out-patient setting as well as another post-prophylaxis study.

Appili recently signed a development and commercialization agreement with Dr. Reddy’s, Global Response Aid (GRA) and Fujifilm.

“With this partnership, we're leveraging not just our studies, but also the Japanese Phase 3 study that Fujifilm just completed and the study in Kuwait that GRA is running.”

All this activity also benefit’s the drug’s safety database, Balboni adds, highlighting they have access to safety data from thousands of patients.

Impressive (H)CV

Tying back to this year’s Nobel Prize, Enanta Pharmaceuticals cut its teeth on HCV.

“Hep C was our cornerstone,” says CEO Jay Luly. “Hep C is what got us into both the liver disease area and what got us into virology area, because it obviously checks both boxes.”

Fighting alongside Vertex and Merck, at the time, the private under-capitalized company knew that it might not be the first to market, so it aimed to be best-in-class, and this is exactly where Luly believes Enanta is with protease inhibitor glecaprevir.

“We teamed up with Abbvie on that, and it was a very successful collaboration that brought two different drugs to market,” says Luly, describing Mavyret, which also includes the antiviral NS5A inhibitor pibrentasvir.

“Not only did we get rid of all those other drugs that weren't very effective and had a lot of side effects, but we took the treatment down from 72 weeks to 52 to 48 to 24 to 12 weeks, and then finally Abbvie and Enanta took it down to eight weeks,” he presses.

Not content, however, Enanta continued to work on its HCV pipeline, developing its own NS5A inhibitor that became a deal with Novartis, nucleotide and non-nucleotide polymerase inhibitors, and they even explored inhibitors of the host protein cyclophilin.

Sticking with the liver, the company is also looking at hepatitis B virus, although ironically, a vaccine exists, often given to children and advised for adults traveling to certain parts of the world.

That said, Luly presses, there are no treatments currently and the vaccine doesn’t help the 250 million people globally who are infected.

“They're on a course that will go from liver inflammation to fibrosis to cirrhosis to transplant and/or liver failure and also hepatic cell carcinoma,” he explains.

In August, at the EASL meeting, Enanta presented the results of its Phase 1 study of EDP-514 versus HBV. The core inhibitor or capsid assembly modulator interferes with viral replication by disrupting the formation of covalently closed circular DNA in hepatocyte nuclei, a hallmark of chronic HBV infection.

In animal models, the candidate has been shown to significantly reduce viral load. It has also shown synergies with nucleos(t)ide reverse-transcriptase inhibitors (NRTIs).

“We and others are hoping to come up a functional cure for hep B, where you can have finite therapy with a durable effect instead of lifelong therapy on [nucleos(t)ide analogues],” Luly says.

While continuing to press on liver diseases, Enanta also turned back to its experiences developing antibiotics against bacterial pneumonia, initiating antiviral efforts in respiratory infections.

In October 2019, the company reported on its Phase 2a human challenge study of N-protein inhibitor EDP-938 in treating respiratory syncytial virus (RSV) infection. After infecting healthy volunteers with RSV, the researchers then treated the subjects with daily or twice daily inhibitor or placebo.

Both doses significantly reduced viral load, as well as nasal mucus production and symptom score.

The trial continues through Phase 2b, and the company expects to initiate two more Phase 2 studies, one in pediatrics and the other in adult stem cell recipients.

“Then we started thinking, well, what are the important other respiratory viruses,” Luly says. “We thought, maybe flu’s reasonably well addressed, so let's look for ones that aren't well addressed.”

Quickly, he continues, they found human metapneumovirus (hMPV), which presents much like RSV.

“Some people have never heard of it before, but if you hang around the [infectious disease] meetings, especially looking at respiratory virus research, you'll see that in many ways hMPV rivals RSV in terms of how many cases there are,” Luly explains.

The company is currently optimizing numerous small-molecule inhibitors with nanomolar EC50, in search of a solid preclinical candidate.

Ironically, in looking to further expand their respiratory portfolio, the company had almost passed on the coronaviruses.

“The original SARS burned out. MERS was very constrained geographically. And the common cold coronas are generally pretty mild,” quips Luly. “And then SARS-CoV-2 came.”

In March, Enanta announced its entry into the SARS-CoV-2 race, taking a two-pronged approach looking at its existing antiviral library for possible leads, but also initiating a program leveraging its direct-acting antiviral expertise to identify novel candidates.

Whereas companies like Enanta, Appili and Cocrystal have focused their attentions on the invading virus, however, other groups have instead homed in on the host machinery that the viruses need to survive.

Targeting the host

For its part, Clear Creek Bio is targeting DHODH, an enzyme at the heart of pyrimidine synthesis in human cells—and thus vital to RNA transcription and DNA replication. And because viruses don’t carry their own supply of pyrimidines, that same pool is vital for their replication, as well.

“Under normal conditions, nucleotides are supplied via both de novo biosynthesis and salvage pathways, the latter of which is a way of recycling pre-existing nucleosides from food or other nutrition,” explained Wuhan University’s Ke Xu and colleagues in a recent paper. “However, in virus-infected cells, a large intracellular nucleotide pool is demanded by rapid viral replication.”

“It is therefore reasonable that de novo nucleotides biosynthesis rather than salvage pathway is more critical for virus replication,” they suggested.

Given the central importance of DHODH, the researchers tested the impact of a series of DHODH inhibitors, including brequinar—the molecule being developed at Clear Creek Bio—on the infectivity of numerous RNA viruses. Not only did these compounds show strong activity against SARS-CoV-2 infection, but also against Ebola, Zika and influenza.

Furthermore, as two of the drugs in this category—leflunomide and teriflunomide—are also indicated for autoimmune diseases, the researchers tested other DHODH inhibitors for activity against cytokine storm, a late-stage manifestation of infection.

They found that when given to infected mice, the DHODH inhibitors significantly reduced the levels of many inflammatory cytokines.

Thus, “DHODHi are effective in infected animals not only by inhibiting virus replication but also by eliminating excessive cytokine/chemokine storm, which suggests the usage of DHODHi could be beneficial to the advanced stage of disease at late-phase infection,” the authors suggested.

Interestingly, the researchers also examined the essential role of DHODH in viral replication by using CRISPR/Cas9 to knock out the gene in cells.

“Unexpectedly, the cell proliferation rate was barely affected in DHODH−/− cells, indicating DHODH is not indispensable for cell growth at least for three days,” the authors noted. “By contrast, as compared to wild-type A549 cells, virus growth was largely inhibited in DHODH−/− cells with a 132-fold reduction of infectious particles at 72 h post-infection.”

The ability of cells to find other sources of pyrimidine synthesis may help explain the safety data of inhibitors like brequinar.

Much like viruses, cancer cells also have a high demand for pyrimidines, and so, as Kumar explains, brequinar was originally developed as a potential cancer treatment in the early 1980s by David Hesson, who was then at DuPont Pharma and is now vice president of development at Clear Creek Bio. Over the intervening decades, Kumar explains, much was learned about its efficacy, dose and schedule.

“The beauty is that we have over 1,000 subjects’ worth of data,” he enthuses.

Thus, he adds, when the company initiated its Phase 1 clinical trial in hospitalized COVID-19 patients in September, it was by no means the drug’s first clinical trial.

In late November, the company dosed the first non-hospitalized CVID-19 patients in a Phase 2 study of brequinar in an outpatient setting.

And much as Appili came to treating COVID-19 indirectly, so too did Selva Therapeutics, which had first licensed in its primary lead SLV213 to treat the parasitic disorder Chagas disease, which has recently started spreading into the United States from Central and South America.

“What really interested us in this asset was an opportunity to address a growing commercial market in the U.S., as well as to obtain a priority review voucher because it's considered a neglected tropical disease,” explains CEO Ted Daley. “But also, we saw an opportunity from a global public health perspective to bring a new and needed better treatment to a lot of people and parts of the world where this disease is more common.”

That parasitic focus is an important part of the story, according to Chief Scientific Officer Felix Frueh, as the company was initially looking for a compound that targeted the parasitic cysteine protease cruzipain. As human cells also have cysteine proteases, there were initial worries that their candidate might have off-target effects.

“In a twist of this story, the off-target effect that we were worried about has become the on-target effect that we now want as the antiviral drugs,” he explains.

Cathepsin L is a cysteine protease found in cellular endosomes, which capture and destroy foreign invaders like virus particles. SARS-CoV-2 and several other viruses, however, harness cathepsin L to its advantage, using its protease activity to help it enter cells.

Thus, rather than increase the likelihood of side effects, SLV213 blocks the activity of cathepsin L and thereby blocks viral entry.

Recently, UC San Diego’s James McKerrow and collaborations, including Selva’s Frueh, evaluated the impact of SLV213 (aka K777) on SARS-CoV-2 infection in both monkey and human cell lines. They found that SLV213 inhibited not only SARS-CoV-2 infection, but also SARS-CoV-1 and MERS-CoV.

In vitro assays showed that SLV213 did not inhibit viral papain-like protease (PLpro) or 3CLpro, but showed potent activity against human cathepsin L, forming irreversible covalent adducts. Proteomic analysis showed that the candidate also targeted cathepsin B, but that only cathepsin L could activate the SARS-CoV-2 spike protein.
An important point that Frueh raises from this study is that even though cells do not express cathepsin L to the same level, Selva has yet to find cells in which SLV213 was not effective.

“It worked to different degrees,” he says. “But if you compare that with several other antivirals and small molecules, it's actually quite astonishing that this seems to really hit a weak spot of the virus.”

In July, the Selva Therapeutics closed a $3-million Series A financing round to help push SLV213 into the clinic. And in November, the FDA cleared its IND application, and the company dosed the first subjects in a Phase 1 clinical trial.

“Our clinical development plan right now is initially focused on treating infected patients,” says Daley. “The bar to initiate and complete a clinical study for SLV213 is lower if we're going into infected patients as opposed to healthy individuals in a prophylactic setting.”

“Having said that, if this is shown to be effective as a treatment, it could be very valuable as a prophylactic for people who are at-risk of exposure to the virus,” he continues.

Daley is quick to point out that SLV213 is the only candidate targeting cathepsin L, which Frueh suggests may also make it an ideal candidate to exploit synergies with other antivirals that might target viral proteases or compounds like remdesivir, which targets the RNA polymerase.

Further in support of Selva’s decision to target cathepsin L comes research from Harvard’s Dennis Kaspar and collaborators to dissect transcriptional and proteomic insights from tissue samples of several patients in Wuhan who died from COVID-19. Despite seeing altered regulation of several transcripts related to cytokines and chemokines, they found little evidence of virus in the tissues. As well, many similar changes were identified in colon tissues that showed no obvious pathogenesis.

The researchers also noted that the levels of serine protease TMPRSS2 were down-regulated in lung, unlike cathepsins B and L, which were elevated.

“Our findings of a low viral burden at the time of fatal outcome may help explain why direct antivirals, such as remdesivir, are more effective in patients with less severe disease, and dexamethasone, an immunosuppressant, is associated with reduced mortality in severely ill patients late in disease,” the authors suggested. “Moreover, the finding that expression of cathepsins B and L—but not TMPRSS2—is elevated in the lungs suggests that inhibitors of these proteases may help prevent viral entry.”

Why choose?

Virus-targeted or host-targeted strategies aside, almost everyone agrees that any anti-infective regimen will involve a cocktail of antivirals or possibly a combination of antivirals and immunotherapies.

“You have a couple of reasons for the use of cocktails,” says Enanta’s Luly. “One is to try to maximize efficacy. And the other is to minimize resistance.”

And as has been seen in the treatment of HIV and HCV, drug combinations are constantly evolving, as older generation drugs that may have debilitating side effects or be expensive are replaced by newer generations of antivirals that are safer, more tolerable and more effective.

Having multiple options may also safe-guard us from future challenges.

“The world is spending too many resources right now if we're only trying to fix COVID-19,” says Clear Creek Bio’s Kumar. “The point of a pandemic preparedness company is that we need to look beyond for the next pandemic, too. We want to be ready for COVID-25, COVID-35.”

A cabinet of broad-spectrum antivirals targeting both the virus and the host would certainly help, especially as we await vaccine development, approval and distribution.

Clique of a mouse

Although the animal origin of SARS-CoV-2 has not yet been fully elucidated, there is no doubt that this was a zoonotic transmission from some animal species to humans. And yet, despite this transmission, the virus remains significantly species-specific, making it difficult to construct animal models of infection and disease. And the original culprits are often no help.

“It's not feasible to take a bat that is infected by SARS-CoV-2 because they're not an amenable species to look for therapies,” says Terina Martinez, field application specialist at Taconic Bioscience. “They just have it. To them, it's like a cold.”

“So, what we're talking about is finding that essential animal model that is susceptible to the virus but gets a pathology that's reasonably translatable to the human condition,” she continues, “so that you can meaningfully study mechanisms and targeted therapies.”

Unfortunately, mice in their native state cannot be infected by SARS-CoV-2. Their version of the ACE2 receptor, the viral spike protein target, is sufficiently different from the human ACE2 to permit binding.

To surmount this challenge, researchers turn to one of two basic methods: viral evolution and transgenesis.

Viral evolution involves adapting the virus to encourage it to bind mouse ACE2. This can be accomplished either by reverse genetic engineering or by passaging the virus through mouse lung tissues to accelerate evolutionary pressures for mouse-adapted sub-strains.

Transgenesis involves inserting the human ACE2 gene into the mouse genome, making the rodent more susceptible to SARS-CoV-2 infection.

In October, Taconic launched the AC70 mouse, which used random transgenesis to insert the human ACE2, and was interestingly an echo of SARS coronavirus past.

“This is a line that was developed Kent Tseng at University of Texas Medical Branch Galveston,” Martinez explains. “It was a model that was developed for the first SARS-CoV.”

“SARS-CoV has about 80-percent shared identity with SARS-CoV-2, and they both use the ACE2 receptor to bind to the human host target cells and integrate into the cells,” she adds.

As the transgenesis was random, however, there may be multiple copy numbers of the human ACE2 and the orientation and location of the insertion could impact gene expression. As well, the murine ACE2 remains intact.

To reduce this uncertainty, other researchers have taken a more targeted approach, replacing the murine ACE2 with its human counterpart. This preserves the regulatory elements of the mouse gene. Unfortunately, says Martinez, these models are not yet widely available.

Although it is good to have multiple options, the specific choice of a given model largely depends on the research question.
“If you're trying to study a therapy designed to inhibit the spike protein on the SARS-CoV-2 virus, then you need to have that human receptor, because it's a very specific interaction,” Martinez explains.

“If what you're studying is a vaccine that causes an immune response that protects against SARS-CoV-2 infection,” she presses, “then you may be able to get away with using a mouse-adapted version that leads to a coronavirus-induced illness that the immune cells can recognize.”

“It may be possible, depending on each institution individual’s biosecurity situation, that the manipulated viruses are at a health status and a biosafety level that's lower than what's required for these really infectious clinical isolates.”

Beyond infection itself, there is also the challenge of how well the pathophysiology of infection in mice matches COVID-19 in humans.

Here again, work from the original SARS and later MERS outbreaks offers assistance as researchers developed models for pathologies like acute respiratory distress syndrome.

Martinez acknowledges, however, that mice and humans manifest disease in different ways, and it may be more a matter of understanding those differences to see the bigger patterns. Weight loss, for example, may signal disease onset long before anyone performs lung histology. And body temperature drops when mice are infected, unlike their human counterparts who develop fever.

“You just need to know what the signs are in the mouse that are your proxy for disease, even if they're not the exact same sign that the human has,” she concludes. “And then there's also overlap when it comes to the severe pneumonia. You could look at inflammatory cytokine as in-life biomarkers before the disease is severe.”

And beyond drug efficacy screening, Martinez also sees an opportunity to use mouse models to identify vaccine-related adverse events, such as those that signal immune hyper-reactivity. In particular, she points to antibody-dependent enhancement, where the immunized body becomes primed to react badly to subsequent infection, and vaccine-associated enhanced respiratory disease, a form of severe pulmonary inflammation first noted in infants vaccinated against respiratory syncytial virus in the 1960s.

Thus, despite the technical challenges of attempting controlled zoonotic transmission, animal models of infectious disease continued to facilitate drug and vaccine development.