In the summer of 1347, the Black Death arrived in Constantinople. “So incurable was the evil, that neither any regularity of life, nor any bodily strength could resist it,” wrote Emperor Ioannes Canta. “Strong and weak bodies were all similarly carried away, and those best cared for died in the same manner as the poor” (1). Before long, the emperor’s youngest son died, along with more than 50 million others as the plague swept through Europe, North Africa, and the Middle East (2).

Miraculously, however, many afflicted with this horrible disease managed to survive. Undoubtedly, the factors that determined whether an infected individual lived or died are complex and varied, but one important factor seemed to be a person’s genetic makeup. In 2022, researchers used ancient DNA to identify a gene variant that associated with increased plague survival and indeed, scientists found that human macrophages with more copies of this gene variant better controlled intracellular replication of plague-causing bacteria (3).

Fortunately, the plague is no longer the scourge it once was, although a few thousand cases still occur each year. Similar to historical plague victims, however, genetics continue to play an important role in infection outcomes for humans today. Now, epidemiologists, geneticists, and microbiologists are working together to identify genes that make humans more or less susceptible to specific diseases. By understanding these genes and their downstream functions, researchers hope to one day leverage this data to develop new strategies to treat or prevent the infectious diseases that still torment humanity.

Patterns of susceptibility

Although it has received relatively little attention, the idea that inherited factors underlie susceptibility or resistance to infectious disease is not an especially new concept. It predates genetic sequencing and even the understanding of DNA’s physical structure. At the dawn of the 20^th century, botanists were already exploring how wheat could be bred to resist a fungal disease called yellow rust and had determined that this characteristic followed Mendel’s laws of inheritance (4).

Over the following decades, researchers began to study this phenomenon first in laboratory animals and then in humans. While studies of infection susceptibility in related individuals — especially comparisons of identical and fraternal twins — strongly suggested that there was a genetic factor at play, for many years, the details of how exactly host genes influence infectious disease processes remained unknown (5).

Evolution has already done these experiments, and we have these variants that are protective. 
- Ellen Leffler, the University of Utah

In recent decades, however, improvements in genetic sequencing have enabled scientists to identify specific genetic variations, both common and rare, that contribute to susceptibility in myriad infectious diseases, including HIV, malaria, typhoid, and influenza (5).

Some researchers, including Jean-Laurent Casanova, a biologist at the Rockefeller University, search for rare mutations by studying individuals, often children, who are especially susceptible to normally benign microbes. For example, researchers noticed that the bacillus Calmette-Guérin (BCG) vaccine, which was normally very safe, was fatal for a tiny fraction of children, fewer than one per million children vaccinated.

The BCG vaccine contains a live but weakened form of Mycobacterium bovis, which protects against the closely related Mycobacterium tuberculosis. In vanishingly rare but tragic instances, otherwise relatively healthy children developed a severe infection with the BCG bacteria after immunization (6). While case reports of these severe BCG infections were published as early as the 1960s, the underlying cause remained a mystery for decades (7). In a 1996 paper, Casanova and his colleagues hypothesized that “the infection probably results from an as yet unknown genetically determined immunodeficiency condition.”

Around the same time, an Imperial College London research group identified clusters of related children, including four from a village in Malta, who seemed especially susceptible to other mycobacterial infections that do not typically affect immunocompetent humans, supporting the involvement of host genetics (8). Further studies by both Casanova’s group and the London group converged on a similar mechanism: mutations in the gene coding for interferon-γ receptor 1 (9,10). Casanova’s group identified a mutation in this gene in a French child who had died from BCG infection, and the London group identified a different mutation in this same gene in the children from Malta.

Since then, Casanova and others have identified mutations in at least 20 genes in individuals with unusual mycobacterial susceptibility, affecting proteins such as the cytokine interleukin-12 and its receptor, as well as the transcription factors STAT1 and IRF1 (11,12). Many of these mutations represent different links in the same chain: involvement in the production of or response to interferon-γ.

The discovery of these mutations provided strategies for the treatment and management of these patients, such as treatment with interferon-γ, which has proven effective in some cases (13). These studies might benefit the larger population as well. By shedding light on pathways and proteins that are critical for overcoming mycobacterial infections, this research could help scientists understand a much more common mycobacterium: Mycobacterium tuberculosis, an infectious agent that is currently the 13^th leading cause of death globally (14).

Since only about five to ten percent of people infected with the bacteria eventually go on to develop disease, identifying important factors in human defenses against mycobacteria might help determine what’s going wrong in people who become symptomatic (14). This is especially relevant as the BCG vaccine is only partially protective and the prevalence of multidrug resistant tuberculosis is on the rise.

From sickle cell trait to hybrid genes

While researchers like Casanova study mutations that confer unusual susceptibility to common infections, some researchers have taken the opposite approach. They use large-scale studies to search for individuals that are unusually healthy despite frequent exposures to disease-causing microbes. One of the first major discoveries in this field came in 1954, when Anthony Allison, then a geneticist at the Radcliffe Infirmary, published his finding that individuals with one copy of the sickle cell gene were somewhat protected from malaria (15).

Malaria affects hundreds of thousands of people each year and, like tuberculosis, available vaccines provide only partial protection and drug resistance is emerging. Some researchers believe that identifying genetic mutations that confer resistance may provide important new avenues for treatment.

“What really drives me is this idea that evolution has already done these experiments, and we have these variants that are protective,” said Ellen Leffler, a population geneticist at the University of Utah. “If we can understand how they work, we hope it will give us more insight. Somehow these genetic variants are able to protect people even though the parasites quickly evolve resistance to our drugs.”

In 2015, the Malaria Genomic Epidemiology Network, which included Leffler, used a genome-wide association study (GWAS) to identify a region on chromosome 4 that related to malaria susceptibility (16). Leffler and her colleagues decided to examine this region more closely, using a reference panel that was more representative of the sub-Saharan African population they were studying. The data pointed to a region of the genome that included the genes for glycophorin A and glycophorin B (17). Glycophorins are structures made of sugars and proteins that are expressed on the surfaces of red blood cells. The glycophorin genes are highly variable throughout the population.

“Glycophorin A and B are two of the receptors that the parasite uses to invade the red blood cell,” said Leffler. “It's been long thought that the variation that we see in human populations could be related to different susceptibilities to malaria parasites. But it’s complicated because the interactions are not essential. We know that if you knock those out that the parasite can still invade.”

Despite not being essential for the parasite’s entry into red blood cells, it appeared that there was still something protective about certain variations in these genes. To investigate further, researchers examined copy number variations in this section of the genome. While most individuals have three glycophorin genes — glycophorins A, B, and E, the last of which is not translated into a protein — the researchers discovered that some individuals had five glycophorin genes (17). These people had two copies of the noncoding E gene, one copy of the A gene, and two copies of a hybrid gene that contained sections from both the A gene and the B gene. These hybrid genes also resulted in hybrid proteins: an intracellular domain that resembled glycophorin A and an extracellular domain that resembled glycophorin B, plus a tiny fragment of A. This genetic variation reduces the risk of severe malaria by 40 percent.

Follow up studies carried out by researchers in Kenya and the United Kingdom demonstrated that this genotype likely confers protection by increasing the tension of the red blood cell membrane, thus making it more difficult for the parasite to invade the cell (18). This suggests that interventions that target red blood cell membrane tension may be helpful in preventing severe malaria.

These results, however, need to be confirmed in the context of the human body, not just cells in a dish. In order to determine just what this genetic variant is doing, said Leffler, “We’re doing some studies of asymptomatic carriers to try to see whether it's protecting against parasite invasion and growth, or if it's just protecting against the symptoms.” Either way, however, this genetic variant could inform new therapies for preventing severe disease.

A pandemic in a tube

GWAS that link genetic variation to disease outcomes in human patients provide important,  but also messy, data. These studies can’t necessarily control for variation in the pathogen or the environment, and so, said Duke University molecular geneticist Dennis Ko, “GWAS of infectious disease has really only happened extensively for a handful of human diseases because you need lots and lots of people infected with the disease for this strategy to really work.”

Ko wanted to develop a method that could be used to study any pathogen, not just the most common ones. So, he created a method called high-throughput human in vitro susceptibility testing, or Hi-HOST, which is essentially a GWAS at the cellular level. Ko used this technique to examine interindividual variation in the response to Salmonella enterica serovar Typhi (S. Typhi), the pathogen that causes typhoid fever (19). Ko and his team obtained immortalized B cells derived from a diverse group of individuals from North America, Europe, Asia, Africa and infected the cells with the typhoid bacteria.

Just like humans experience different severities of typhoid infections, the cells showed differences in their abilities to control the intracellular replication of the typhoid bacteria. Researchers searched for locations in the cells’ genomes that associated with differences in pathogen replication. Only one region was significant: a stretch of DNA on chromosome 1. They discovered that this polymorphism affected the expression of a mucolipin gene called MCOLN2. The allele that resulted in less MCOLN2  expression also associated with greater bacterial replication. To test whether this gene really was important, researchers eliminated MCOLN2  expression in a group of human cells and then exposed them to the typhoid bacteria.

“Oftentimes when we knock down a gene, we'll get no effect, or we'll get a small effect,” said Ko. “This time, we got the biggest phenotype we've ever seen.” Just 24 hours postinfection, bacterial replication increased by 2.5 fold in cells without MCOLN2.

However, it wasn’t clear just how human MCOLN2 — an ion channel that sits in the membranes of vacuoles within cells — restricted bacterial growth.

“We let the salmonella tell us the answer,” said Ko. Using transcriptomics, researchers eavesdropped on salmonella bacteria in human cells with and without MCOLN2. “When this mucolipin channel is present, the salmonella transcriptome looks like it's starved for magnesium,” said Ko. The team then confirmed that mucolipin could serve as a magnesium channel, helping the human cell sequester this essential nutrient away from bacterial cells, putting the brakes on bacterial growth.

We let the salmonella tell us the answer. 
- Dennis Ko, Duke University

More recently, Ko and his research team have developed an updated version of Hi-HOST called scHi-HOST. “We can put 500 different cell lines in the same tube and expose them all to the same virus,” said Ko. “It’s like a pandemic in a single tube.” The team then uses single-cell RNA sequencing to determine the identities of the cells, considerably streamlining the process.

So far, Ko’s team has used this new technique to identify a mutation in the ERAP1 gene that increases influenza susceptibility. The team is now exploring how to apply this method to a variety of other viruses, including Zika and pox viruses (20).

Studying human genetic variation in this context might also help scientists respond to future infectious threats. For example, said Ko, “If we see that there's an emerging flu strain in human populations, we can rapidly test with scHi-HOST and determine if the cellular response differs from the other flu strains or if we can measure particular susceptibility or resistance.” This might help researchers identify people at high risk or suggest potential therapies even before the pathogen becomes widespread.

Genetic variant-inspired therapies

For some diseases, scientists are only just beginning to identify protective mutations, while for other diseases, these mutations have already informed treatments that have demonstrated preliminary success. One instance is HIV.

In 1996, only 13 years after the virus was first isolated, three different research groups identified a mutation that conferred protection against macrophage-tropic strains of HIV (21). This mutation, named CCR5Δ32, was a deletion of 32 base pairs in the gene coding for CCR5, a coreceptor that these strains of the virus needed to enter cells.

Barely a decade later in 2007, stem cells from a donor with the CCR5Δ32 mutation were used in the first instance of an HIV cure (22). Timothy Ray Brown, known for many years as the Berlin patient, was diagnosed with HIV in 1995 and with acute myeloid leukemia in 2006. As a treatment for leukemia, Brown received a bone marrow transplant, but researchers ensured that the donor cells contained this HIV-protective mutation. As they hoped, Brown’s levels of HIV became undetectable in blood and biopsies, even though he was no longer taking antiretroviral therapy. While this curative method has been replicated in a few other patients, bone marrow transplants come with significant safety risks and are resource intensive, so this is not a feasible cure for the majority of patients. Nevertheless, it provides a proof of concept for researchers seeking to develop other cures using the same principles.

Meanwhile, Howard Gendelman, now an infectious disease and pharmacology researcher at the University of Nebraska Medical Center, was studying Visna virus, a lentivirus that causes a multiple sclerosis-like condition in sheep at Johns Hopkins University in the early 1980s, when HIV, another lentivirus, began spreading throughout the United States.

“I had seen some of the early patients when I was in New York as a resident,” said Gendelman. “I knew what the disease was and that it was becoming a big problem.” So, when Anthony Fauci, then the director of the National Institutes of Health’s National Institute of Allergy and Infectious Diseases, invited him to apply his lentivirus expertise to studying HIV at the NIH, Gendelman couldn’t refuse.

Gendelman has been studying HIV ever since, including developing long-acting antiretroviral therapies (23). While antiretroviral drugs suppress viral replication and reduce viral load and have been game changing for the treatment of HIV, they do not cure the disease. HIV inserts its own DNA into the host DNA and resumes replication as soon as the drugs wear off.

To solve this problem, Gendelman teamed up with Kamel Khalili, a neurovirologist at Temple University who had developed a gene-editing strategy to remove HIV DNA from the host genome. This has been a productive partnership: “When people have a common enemy — in this case, HIV — it really brings us together and sets up an environment of camaraderie,” said Gendelman.

When people have a common enemy — in this case, HIV — it really brings us together and sets up an environment of camaraderie. 
- Howard Gendelman, the University of Nebraska Medical Center

In 2019, the team combined long-acting antiretroviral therapy with a CRISPR-based treatment to excise the HIV viral DNA from the genomes of mice with human immune cells. The treatment was successful, but only in some animals; it completely eliminated the virus in about 30 percent of the treated mice (24).

The team wanted a better treatment response, so they combined these two treatments with another CRISPR-based therapy to inactivate CCR5, mimicking the CCR5Δ32 mutation. With this addition, said Gendelman, “any residual virus that's still present after the antiretroviral therapy will be blocked from infecting any new cells.” Their hypothesis proved correct. This approach cured approximately twice as many mice, indicating that targeting CCR5 may be an important consideration for future trials seeking to cure HIV with gene therapy (25).

While many infectious disease researchers focus on the properties of pathogens to understand the outcomes of infections and suggest new treatments, in some cases, human genetic variation might be equally important. From tuberculosis to HIV, malaria to influenza, human genetic variation provides important insights into major causes of human disability and death.

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