Characterization of Novel and Diverse Henipavirus Glycoproteins

Abstract

The Henipavirus genus includes the deadly Nipah and Hendra viruses, both zoonotic BSL4 pathogens that can cause infection fatality rates of over 50%. These viruses were first identified in the 1990s after being responsible for deaths in Australia and Malaysia, with the Nipah outbreak in Malaysia being wider in scope, resulting in over 100 deaths and requiring the culling of over 1 million pigs to contain the spread of disease. Since then, Nipah virus has continued to periodically reemerge, mostly in Bangladesh and India. While these subsequent outbreaks have fortunately been of small scale, fatalities still occur due to the extreme lethality of Nipah virus.The primary hosts for Nipah and Hendra are fruit bats of the Pteropus genus. They can easily infect a range of additional mammals such as horses and pigs, enabling the frequency of zoonotic outbreaks of disease in humans and ensuring a continuing threat of infection given the overlap of fruit bat and human habitation. There are currently no approved vaccines or therapies to treat Henipavirus infection. The combination of virulence, lack of countermeasures, and ease of zoonotic transmission underscores the pandemic potential of the Henipaviruses and as a result, they are on the WHO list of priority pathogens. As with all other members of the Paramyxoviridae family, Henipaviruses are enveloped viruses that rely on a viral fusion protein to facilitate entry into host cells. They specifically utilize a class I fusion protein, a class of surface glycoprotein widely used by viruses, with examples including the HIV envelope protein (Env), Influenza hemagglutinin protein (HA), and Coronavirus Spike protein (S). There are important mechanistic similarities between all class I fusion proteins, but the Paramyxoviruses differ significantly from other examples in that they separate attachment and fusion roles into separate glycoproteins. These proteins operate together to facilitate fusion, but knowledge of many key details of their interactions both with the host cell and with each other is lacking. Given that they are the sole surface proteins on Henipavirus virions, they are the primary targets for developing vaccines and treatments, and essential to this translation into therapies is a deeper understanding of their structure, mechanisms, and antigenicity, so that sites of vulnerability can be identified. Since the initial discovery of Nipah and Hendra virus, additional members of the genus have been discovered, with Langya virus, discovered in China in 2022, resulting in symptomatic human infections. Surveillance efforts are continuing, with new species being discovered in additional mammalian hosts over a wide geographic range. Structural and antigenic characterizations of Henipaviruses to date have focused primarily on Nipah and Hendra virus. To be prepared for the emergence of future infectious Henipaviruses, it is important to study these emergent species and identify mechanistic and antigenic trends across this expanding group of pathogens. This dissertation characterizes biochemical, biophysical, and antigenic properties of dozens of Henipavirus species which, until now, were only known through sequence, setting a foundation for future vaccine and therapeutic development. It details the process of identifying, classifying, and producing a panel of Henipavirus glycoproteins with unique amino acid sequences that can be used to study the current breadth of the genus. It reports the results of an antigenic analysis that both establishes similarities and boundaries within the genus and details several novel antibodies with extremely broad reactivity. Additionally, this dissertation compiles multiple studies that generated atomic-resolution structures of novel Henipaviruses, revealing new insights into the mechanisms of Henipavirus fusion proteins. These structures served as the basis for development of effective and translatable strategies for controlling the conformational state of the fusion protein, a valuable tool for vaccine immunogen design. Finally, this dissertation details the method development process for a fluorimetry-based assay that can identify the conformational state of Henipavirus fusion proteins quickly and with minimal sample usage, bypassing more lengthy structural assays and establishing a means for high throughput evaluation of conformational stabilization efforts. This work is effectively the first biochemical characterization of any kind for the glycoproteins of most of the recently identified Henipavirus species. It details trends, similarities, and distinctions that can inform future drug development, including outlining antigenic boundaries that may serve as realistic targets when considering countermeasures designed to have broad potency. The mechanistic insights from structural studies and newly developed assays will aid in future Henipavirus studies that aim to increase pandemic preparedness.