Interference with Virus Infection

In eukaryotes, type 1 IFN is essential for defense against virus infection and is a major component of antimicrobial defenses and the innate immune response. The discovery of IFN was driven by the new field of virology (1) and studies of influenza A virus (IAV) in the 1950s through which the interferon was defined by Alick Isaacs and Jean Lindenmann. These investigators revealed IFN as a soluble agent that was made from infected cells and could suppress IAV infection (2, 3). The discovery of IFN serves as a cornerstone from which the foundation for the discipline of innate immunity developed within the field of immunology.

Today, we know that IFNs make up an important family of cytokines that have antimicrobial, proapoptotic, immunomodulatory, and antiproliferative actions. There are three types of IFN, each encoded by unique gene(s) and named based on their order of discovery and related biology. The major type 1 IFNs include IFN-α and IFN-β. In humans, there are multiple IFN-α genes encoding highly related, but unique, IFN-α subtypes and a single IFN-β gene, all clustered on chromosome 9 (4). Each can be expressed from most nucleated cells of the body and encodes a protein that binds to a common IFN-α/β receptor. The type 1 IFNs also include minor subtypes IFN-ω, IFN-τ, and IFN-κ. These IFNs were discovered after IFN-α/β, are categorized as type 1 IFNs, and are selectively expressed among cell types and species (4). IFN-γ is the sole type 2 IFN and is encoded by a single gene expressed by activated immune cells. IFN-γ binds to the IFN-γR expressed by many cell types, including immune cells and tissue parenchyma cells. Type 3 IFNs are now known as IFN-λ and, in humans, include three subtypes with a receptor chain combination that is selectively coexpressed by myeloid cells and by parenchymal cells of the liver and gut. During virus infection of epithelial cells, IFN-β is typically induced first, followed by signaling events that drive the expression of IFN-α subtypes. Virus infection also induces IFN-λ expression and, in certain cell types, the additional type 1 IFNs can be produced. These actions mark the innate immune response and the first stage of the global immune response to virus infection. In addition to antiviral actions, the IFNs serve to regulate the quality and actions of the adaptive immune response by modulating immune effector cell activation and function (5). Isaacs and Lindenmann discovered what we know as type 1 IFN and described its virus-induced, cell-derived, and soluble nature and defined antiviral actions that interfere with virus infection (2, 3). Hence, they assigned the name “interferon” to what is now known as the IFN family of cytokines.

How did they discover the interferon? At the time, Alick Isaacs was studying the infection properties of IAV and other RNA viruses, including a variety of mosquito-transmitted viruses, such as West Nile virus. Working at the National Institute for Medical Research in London, he met a research fellow named Jean Lindenmann who was learning the ropes in the upstart field of virology. It had just been realized that viruses were different from bacteria in that they replicated through a means beyond binary fission within animal cells. This replication occurred during what the early virologist Leslie Hoyle described as the eclipse phase in his studies of IAV, by which he defined a noninfectious phase of viral growth (6). Indeed, the eclipse phase is a period of viral disassembly and synthesis occurring right after virus entry into the host cell during acute infection, thus reflecting the specialized and parasitic virus life cycle. Additionally, a previous advance spearheading the ability to study viruses in tissue was applied by Isaacs and Lindenmann to culture IAV when they used Thomas Goodpasture’s (7) methods described for the culture of fowlpox virus in chorioallantoic membranes of chicken embryos. Isaacs and other investigators also figured out that one could heat inactivate virus stocks to render them noninfectious while retaining properties of tissue interaction. Using methods of IAV culture in chorioallantoic membranes, Isaacs and Lindenmann produced IAV stocks. They then heat inactivated a set of IAV stocks to use for studies of infection competition between nontreated, infectious IAV and inactivated IAV.

In their first series of experiments, Isaacs and Lindenmann exposed chorioallantoic membranes to inactivated IAV for various times at 4 or 37°C prior to exposure to infectious IAV at 4 or 37°C. These were the primordial days of tissue culture wherein cultures were little more than crude slices of membrane of approximately equal size floating in a glass dish of saline solution in a slightly warmed oven without additional CO₂. In these experiments, the membranes were exposed to inactivated IAV. Heat-inactivated IAV can still be internalized into the target cell at 37°C but not at 4°C, and the membranes were further incubated to allow inactivated virus internalization. Membranes were then washed in saline solution to remove residual IAV particles and placed back in culture for exposure to infectious IAV for 48 h, after which supernatants were collected. Dilutions of supernatants were then mixed with chicken RBCs. IAV can agglutinate RBCs, so the number of IAV particles produced by the infected chorioallantoic membrane culture was determined by assessing heme agglutination activity of the supernatant. These studies revealed that, when conducted at 37°C, a 15-min pre-exposure to inactivated IAV resulted in membrane interference or resistance to infectious virus at 37°C, because only input or lower levels of virus were generated after infectious IAV exposure. In these studies, the peak of interference occurred after a 6-h pre-exposure. Moreover, when pre-exposure was conducted at 4°C, followed by exposure to infectious IAV at 37°C, no interference against infectious IAV was observed, indicating that tissue metabolic events were necessary for viral interference to occur. Thus, a synthetic product from the chorioallantoic membrane was likely mediating the interference against infectious IAV. Further experiments to treat fresh membranes with ground-up membranes or supernatant from membranes that had been exposed to inactivated IAV for 6 h revealed that interference was generated from membrane exposure to a soluble product from the virus-exposed membranes. Thus, the soluble product was named “interferon.”

Isaacs and Lindenmann, with Burke and Valentine (2, 8), conducted follow-up studies to evaluate the properties of IFN. They scaled up their chick embryo chorioallantoic membrane culture system to produce larger quantities of IFN-containing supernatant by exposing membranes to inactivated IAV for 3 h, washing the membranes in saline, and then culturing them for 24 h in fresh media, after which the media was collected as the IFN preparation. This preparation was then used to treat membranes and assess their resistance to infection by Sendai virus and Newcastle disease virus (both are paramyxoviruses related to the contemporary human pathogen respiratory syncytial virus) and to vaccinia virus (a derivative of smallpox virus). In additional studies, Isaacs and Westwood (9) similarly assessed IFN actions on West Nile virus infection. Upon pretreatment of membranes with IFN supernatant, in each of these studies it was found that membranes could resist the virus infection. Moreover, the studies showed that the IFN effect was titrated away upon serial dilution of the IFN supernatant. Thus, IFN actions were dose dependent. It also was determined that IFN was heat labile and was not neutralized by anti-IAV Abs. It was concluded that IFN was produced from infected cells, either as a fragment of the virus that then stimulates interference with new infection or as a reaction product of the cell, such as a cellular enzyme. Isaacs proposed the hypothesis that IFN was a product of the heat-inactivated IAV, in which the inactivated virus feeds into the cell insufficient information for orderly virus replication, instilling a dysregulated viral-replication process that interferes with the infection, and that the IFN that is produced could block viral replication. Isaacs and Lindenmann were absolutely correct.

Today, we understand that Isaacs and Lindenmann had observed the production of type 1 IFN induced by inactivated IAV, similar to how defective virus particles can stimulate IFN production when viral pathogen-associated molecular patterns are revealed during the eclipse phase of the infection and are sensed by pattern recognition receptors in the target cells. Their discovery of IFN, now 58 y ago, led to the identification of other IFNs, the IFNRs, JAK-STAT signaling pathways, IFN regulatory factors, IFN genes, and the pattern recognition receptors, such as TLRs and the RIG-I–like receptors that induce intracellular signaling to activate IFN regulatory factors to drive IFN production and the innate immune response to virus infection. Moreover, this work led to studies that identified plasmacytoid dendritic cells as the major IFN-producing cell and provided the foundation for studies that defined other IFN-producing cell subsets. We know that IFN actions are mediated by hundreds of IFN-stimulated genes and that specific IFN-stimulated gene actions suppress infection by IAV and other viruses. Today, pharmacologic IFNs serve as effective therapeutics for treating virus infection, cancer, and autoimmune disease (10–13). The work of Alick Isaacs and Jean Lindenmann opened the door for these discoveries and products, with great impact on immunology, virology, microbiology, and public health.