Virologist Theodora Hatziioanno “broke the species barrier” and became the first person to develop a non-human or chimp AIDS model in 2014. She’s also been making waves lately because she’s helping lead efforts to study antibody responses in the blood of recovered COVID-19 patients. That’s how I first heard of her, but with a quick Google search I learned about that milestone. Some more digging and I learned about a lot of other cool work she’s done. What didn’t I find? A Wikipedia page. So I fixed that. And now I want to tell you more about her and her work. 

As I mentioned, I first heard of her in the context of coronavirus research. As an Associate Research Professor at the Rockefeller University in NYC, she was at an early epicenter of the COVID-19 pandemic and she quickly sprang into action to develop and put to use ways to test the ability of antibodies present in recovered patients’ blood to block coronavirus infection (and the ability of the virus to escape) – I’ll tell you more about that later. But first I want to tell you about her years of work studying how *HIV* is able to escape species-specific cellular defenses called “restriction factors.” 

HIV stands for Human ImmunoDeficiency Virus and it’s the virus that causes the disease AIDS (Acquired Immune Deficiency Syndrome). According to UNAIDS, 38 million people around the world are infected with HIV, 1.7 million people became newly affected in 2019, and 690,000 people died from AIDS in 2019. First reported in 1981, HIV/AIDS clearly remains a huge global problem. However, while there is no cure (yet) for AIDS, decades of research by scientists has turned HIV from a death sentence into a manageable chronic condition (if treatment is accessible…) ⠀

HIV is a retrovirus. Instead of “retro” in the sense of discos and roller skates, it’s retro in the sense that it “goes back” from RNA to DNA, violating the central dogma of molecular biology that says that genetic information flows from DNA (original instructions) to RNA (recipe copies) to protein. HIV travels with its genome (genetic blueprint) in the form of RNA, encapsulated in a coat of a protein called capsid (CA). Once inside the cell it releases its RNA from the capsid & “reverse transcribes” it into DNA. This allows it to sneak into our genome, which is written in DNA. HIV infects a kind of immune cell called a T cell, and, once integrated it can either hang out and not really do much damage, or it can produce a lot of new virus (through viral replication) that can go infect a lot more T cells. 

One of the things that’s made AIDS hard to study has been a lack of good animal models, in part because it’s been hard to get HIV to infect non-hominids (not humans or chimps). In studying *why* it was so hard to infect them, Hatziioanno (and other groups) discovered a number of cellular defenses called “restriction factors.” Unlike antibodies, which are a form of adaptive immunity (your body learns to make them in response to a virus), restriction factors are a form of “innate immunity” – they’re always there (although some only get made in large amounts once they get some form of stimulation (e.g. by the signaling molecule interferon, which is produced in response to a variety of foreign-thing triggers). 

A few important restriction factors for HIV-1 are TRIM5α, which binds to the capsid once it enters the cell and leads to its degradation; APOBEC3, which extensively mutates the viral DNA so that it becomes gibberish; and tetherin, which is a membrane protein that tethers new virions on the cell surface as they’re trying to escape. 

But HIV has countermeasures…

cell gives you APOBEC3 – HIV gives you Vif, which binds it, calls in ubiquitin ligases to add a destruction tag and sends it for degradation

cell gives you tetherin – HIV gives you Vpu, which binds to tetherin and leads to its degradation.

Your body doesn’t have to “learn” to make restriction factors, but a virus can “learn” to evade them by adapting their counteracting proteins. So, for example, they can change their Vif or Vpu, or change their capsid to evade TRIM5α. This “learning” happens because viruses can replicate rapidly – and make mistakes when they do – so if they make mistakes that make them less susceptible to the restriction factor (but which don’t hurt themselves too much) they’ll be at an advantage. 

Crucially, the virus is going to learn to evade the immune system of its host species, because that’s what it’s feeling the pressure from. So the H in HIV really matters – this virus has adapted to evade human versions of restriction factors. Even though other species have related restriction factors, they’re often different enough that the virus is vulnerable to them. So the virus has a hard time infecting and/or replicating in different species. 

Strains of a related lentivirus (the kind of virus HIV is) called Simian Immunodeficiency Virus (SIV) *can* (and do) infect monkeys and apes, and it’s been used a lot as a model for studying HIV, but it’s really different, and those differences can (and sometimes do) prove critical when it comes to testing out treatments, vaccines, etc. To make the SIV more HIV-like, scientists tried putting some of the HIV genes into SIV to make chimeric viruses called SHIVs, but they still were mostly SIV. Theodora Hatziioanno wanted to try going the other direction – take HIV and make it slightly SIV-like, swap in just enough SIV that it can infect and replicate in a monkey.

Working with her husband and colleague Paul Bieniasz, she started with a commonly-used research monkey: the Rhesus macaque (whoah, I just spelled that right the first time!) 

In a 2006 paper in Science (link at bottom), she showed that if she took HIV-1 and…

  1. replaced the gene for the human-adapted capsid (CA) with the gene for a rhesus-adapted one (SCA, where S is for Simian because it comes from an SIV) so that it could evade the rhesus TRIM5α and
  2. replaced the HIV-1 Vif gene with a rhesus-adapted SIV Vif (SVif)

…she could get the virus to infect and replicate in rhesus cells. She’d had to replace 2 genes, but the virus (which she termed simian tropic HIV-1 (stHIV)) was still 88% “original” HIV-1. This was an important milestone, but the capsid’s pretty important so if she could get something where she didn’t have to tweak that, that’d be way better. 

So she needed to find an animal whose TRIM5a couldn’t bind the HIV-1 capsid. And she did – actually multiple groups found it at the same time and they were published back to back. Turns out both the owl monkey and the pigtail macaque had a mutated TRIM5, termed TRIM5Cyp because it had another gene called Cyp inserted in it. And the pigtail’s TRIM5Cyp couldn’t bind HIV-1 capsid. 

This suggested that HIV-1 could infect pigtail macaque cells – and she found that it could indeed. Problem was, the pigtail macaque cells still had pigtail macaque APOBEC3. APOBEC3 is that RNA mutator (it’s a “cytidine deaminase” that changes the RNA letter C). HIV-1’s Vif protein had adapted to bind human APOBEC3 and trigger its degradation, so the RNA stays safe. But it didn’t recognize the pigtail APOBEC3, so, although the unaltered HIV-1 could infect pigtail cells, it couldn’t replicate because it would have its genome turned into gibberish. 

To “fix” this, she returned to what she’d learned from the rhesus work – she swapped out the HIV-1 Vif gene with a macaque-adapted Vif gene from an SIV (Vifmac) and tried infecting cells. It worked! The virus (which was all HIV-1 except for Vif) infected pigtail lymphocytes (a type of blood cell) and replicated (as they could measure by measuring the viral RNA levels in the growth media the cells were in.

But would it work in actual monkeys? To test this out they collaborated with people who had monkeys – the lab of Jeffrey Lifson at the Frederick National Laboratory. And it worked! Well, kinda… The virus could infect the monkeys and replicate a little, but the monkey quickly squashed it. This wasn’t that surprising because HIV-1 had spent years adapting to humans and it’s not just that one gene that makes humans and pigtails different. So they decided to give the virus some evolutionary time to catch up. They took their virus and “passaged it” through pigtails. First they’d temporarily deplete the immune system of the pigtails by using anti-CD4+ antibodies to remove those important immune cells from the blood for a couple weeks. Then they’d infect a monkey, let the virus replicate and mutate, slowly adapting to better grow in the pigtail, isolate that virus and use it to infect another immune-depleted monkey. 

The first 3 passages didn’t hurt the monkeys much, but at the 4th passage, mutations arose that made it much stronger – strong enough that the viral levels remained high. And, crucially, strong enough that the monkeys showed AIDS-like signs including T cell depletion, tumors, and opportunistic infections including Pneumocystis pneumonia. The monkeys became so sick, in fact, that they had to be euthanized. 

So, for the first time ever, they had a good monkey model in which to study HIV/AIDS! Well… almost – the virus still wasn’t strong enough to sicken monkeys without prior immunodepletion. 

They looked to see what viral changes had occurred between the 3rd & 4th passages, and one of the key ones was mutations in the Vpu gene. That’s the viral counter to the tetherin restriction factor. They found that just a couple opportunistic letter swaps in the Vpu gene had made it better at binding and triggering degradation of tetherin before tetherin could trap it on the cell surface. 

If you want to hear Hatziioanno describe the work in her own words, she talked about it in an interview on This Week in Virology (TWiV 465 )

I had actually first heard Hatziioanno at a (virtual) meeting about her work on SARS-CoV-2 (“the coronavirus”) and COVID-19 (the disease it causes). So I thought I’d tell you about that too…

The work she presented has to do with antibodies, which are little proteins produced by immune cells called B cells, that can bind to specific regions, aka epitopes, of foreign things (antigens) such as viral proteins. If antibodies bind to a virus in such a way that the virus can no longer infect cells, we call it a “neutralizing antibody” or “nAb.” For SARS-CoV-2, nAbs often bind to the Spike protein and block it from binding to the ACE2 receptor. They have significant value both for the patient (as they can provide some protection against reinfection) as well as potential therapeutic use. Therefore, scientists are really interested in seeing if people make them and whether we can isolate the B cells making them and make lots more – and Hatziioannou and Bienesz have been a couple of the scientists who’ve really been leading these efforts in the US.

Hatziioannou has been looking at the blood plasma (the cell-less part of blood) of recovered patients to test for nAbs (unfortunately, in NY there’s no shortage of test material). When analyzing data from hundreds of NY donors, what she found was really interesting; about 80% of recovered patients had detectable nAbs, but the distribution of antibody levels within that 80% of people were highly skewed. Most people only had low levels of nAbs; however, about 10% of people were “elite neutralizers,” churning out large numbers.

Don’t be scared off by her finding that most people only made small amounts of nAbs – quantity isn’t everything, you also have to take into account how well the nAbs can bind and block the virus. In fact, she found that the majority of people produced some really strong ones and she was able to isolate some of the strongest and test their therapeutic potential. 

As we saw above with HIV, every time the virus replicates (copies its genome) it can make little mistakes (mutations). Most of these are harmless (or even detrimental to the virus) but some give the virus beneficial properties under certain selective pressures (like the presence of antibodies). By growing the virus in the presence of different nAbs, Hatziioannou was able to select for viruses that had made mistakes in the gene for the Spike protein which allowed them to resist binding to the nAbs – a phenomenon called “viral escape.” When she sequenced the mutated Spike genes from those viruses, she found that resistance to different nAbs clustered to mutations in different specific locations on Spike. 

She then looked at the sequences of the Spike gene from patient samples and found that, although rare, some indeed had mutations in those regions which would likely make them resistant to individual nAbs. So, if you were to try to treat one of those patients with that nAb, they likely wouldn’t respond. However, she presented evidence that combinations of nAbs targeting different regions (epitopes) of Spike could prevent that “viral escape” as it’s unlikely a virus will have a Spike that has multiple resistance-giving mutations. Therefore, she suggested that for treatment/prophylaxis, multiple nAbs be given as an “antibody cocktail” (this is the strategy being taken by Regeneron).  

Some more about Hatziioannou (some of what you can now find on Wikipedia – and remember you can help me improve her article even more because anyone can edit!): Theodora Hatziioannou was born and raised in Rhodes, Greece. She studied biochemistry at the University of Bristol in Britain and then got a Master’s degree in biotechnology from Imperial College, London. She worked as a research technician with Robin Weiss at the Institute of Cancer Research, where she says she fell in love with research and determined she wanted to go on to earn a PhD. She therefore moved to Lyon-France, where she earned a PhD from the University Claude Bernard in 1999.Her PhD research, carried out under François-Loïc Cosset, involved looking at adapting retroviruses to use as tools for gene therapy, seeking to expand their tropism and target them to specific cells by manipulating the retroviral envelope. After earning her PhD, Hatziioannou moved to the United States, where she joined the lab of Stephen Goff at Columbia University as a postdoctoral fellow. She then did further postdoctoral research with Paul Bieniasz at the Rockefeller University and the Aaron Diamond Research Center for AIDS.She became an Assistant Professor at the Rockefeller University in 2006 and was promoted to Associate Professor in 2012.

here’s that Wikipedia link:  

And you can follow her on Twitter @theodora_nyc

here are those key papers:

If you want to learn more about…

you can find more on coronavirus-y topics here (including some in Greek thanks to Nefeli Boni-Kazantzidou): 

and more explanations about all sorts of things: #365DaysOfScience All (with topics listed) 👉

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