The impact of PA/I38 substitutions and PA polymorphisms on the susceptibility of zoonotic influenza A viruses to baloxavir.

Genetic reassortment of avian, swine, and human influenza A viruses (IAVs) poses potential pandemic risks. Surveillance is important for influenza pandemic preparedness, but the susceptibility of zoonotic IAVs to the cap-dependent endonuclease inhibitor baloxavir acid (BXA) has not been thoroughly researched. Although an amino acid substitution at position 38 in the polymerase acidic protein (PA/I38) in seasonal IAVs reduces BXA susceptibility, PA polymorphisms at position 38 are rarely seen in zoonotic IAVs. Here, we examined the impact of PA/I38 substitutions on the BXA susceptibility of recombinant A(H5N1) viruses. PA mutants that harbored I38T, F, and M were 48.2-, 24.0-, and 15.5-fold less susceptible, respectively, to BXA than wild-type A(H5N1) but were susceptible to the neuraminidase inhibitor oseltamivir acid and the RNA polymerase inhibitor favipiravir. PA mutants exhibited significantly impaired replicative fitness in Madin-Darby canine kidney cells at 24 h postinfection. In addition, in order to investigate new genetic markers for BXA susceptibility, we screened geographically and temporally distinct IAVs isolated worldwide from birds and pigs. The results showed that BXA exhibited antiviral activity against avian and swine viruses with similar levels to seasonal isolates. All viruses tested in the study lacked the PA/I38 substitution and were susceptible to BXA. Isolates harboring amino acid polymorphisms at positions 20, 24, and 37, which have been implicated in the binding of BXA to the PA endonuclease domain, were also susceptible to BXA. These results suggest that monitoring of the PA/I38 substitution in animal-derived influenza viruses is important for preparedness against zoonotic influenza virus outbreaks.

Safety and immunogenicity of a booster dose of S-268019-b: Interim findings of a Phase 3, open-label clinical study in Japan.

Despite the initial success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in prevention of symptomatic and severe diseases, booster vaccination has become increasingly important with the advent of variants with immune-escaping capacity. Herein, we report the safety and immunogenicity of S-268019-b, comprising SARS-CoV-2 spike protein and a squalene-based adjuvant, as a booster dose. We performed an interim analysis of an open-label, Phase 3 study data until Day 29 following S-268019-b booster in Japanese adults (aged 20-64 years) who had completed primary vaccination with mRNA-1273 and in Japanese elderly (aged ≥ 65 years) who had completed primary vaccination with mRNA-1273 or BNT162b2. Reactogenicity was mild in most participants; no serious treatment-related adverse events were noted. S-268019-b enhanced SARS-CoV-2 neutralizing antibodies, immunoglobulin G antibodies, and predominant T-helper 1-mediated immune reaction in all cohorts, regardless of age, in Japanese participants with prior vaccination with mRNA vaccines.

A-910823, a squalene-based emulsion adjuvant, induces T follicular helper cells and humoral immune responses via α-tocopherol component.

Background: Adjuvants are chemical or biological materials that enhance the efficacy of vaccines. A-910823 is a squalene-based emulsion adjuvant used for S-268019-b, a novel vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is currently in clinical development. Published evidence has demonstrated that A-910823 can enhance the induction of neutralizing antibodies against SARS-CoV-2 in humans and animal models. However, the characteristics and mechanisms of the immune responses induced by A-910823 are not yet known. Methods and Results: To characterize A-910823, we compared the adaptive immune response profile enhanced by A-910823 with that of other adjuvants (AddaVax, QS21, aluminum salt-based adjuvants, and empty lipid nanoparticle [eLNP]) in a murine model. Compared with other adjuvants, A-910823 enhanced humoral immune responses to an equal or greater extent following potent T follicular helper (Tfh) and germinal center B (GCB) cell induction, without inducing a strong systemic inflammatory cytokine response. Furthermore, S-268019-b containing A-910823 adjuvant produced similar results even when given as a booster dose following primary administration of a lipid nanoparticle-encapsulated messenger RNA (mRNA-LNP) vaccine. Preparation of modified A-910823 adjuvants to identify which components of A-910823 play a role in driving the adjuvant effect and detailed evaluation of the immunological characteristics induced by each adjuvant showed that the induction of humoral immunity and Tfh and GCB cell induction in A-910823 were dependent on α-tocopherol. Finally, we revealed that the recruitment of inflammatory cells to the draining lymph nodes and induction of serum cytokines and chemokines by A-910823 were also dependent on the α-tocopherol component. Conclusions: This study demonstrates that the novel adjuvant A-910823 is capable of robust Tfh cell induction and humoral immune responses, even when given as a booster dose. The findings also emphasize that α-tocopherol drives the potent Tfh-inducing adjuvant function of A-910823. Overall, our data provide key information that may inform the future production of improved adjuvants.

Results from a preclinical study in rodents and a Phase 1/2, randomized, double-blind, placebo-controlled, parallel-group study of COVID-19 vaccine S-268019-a in Japanese adults.

BACKGROUND: In early 2020, developing vaccines was an urgent need for preventing COVID-19 from a contingency perspective. METHODS: S-268019-a is a recombinant protein-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a modified recombinant spike protein antigen adjuvanted with agatolimod sodium, a Toll-like receptor-9 agonist. In the preclinical phase, it was administered intramuscularly twice at a 2-week interval in 7-week-old mice. Immunogenicity was assessed, and the mice were challenged intranasally with mouse-adapted SARS-CoV-2 at 2 and 8 weeks, respectively, after the second immunization. After confirming the preclinical effect, a Phase 1/2, randomized, parallel-group clinical study was conducted in healthy adults (aged 20-64 years). All participants received 2 intramuscular injections at various combinations of the antigen and the adjuvant (S-910823/agatolimod sodium, in µg: 12.5/250, 25/250, 50/250, 25/500, 50/500, 100/500, 10/500, 100/100, 200/1000) or placebo (saline) in an equivalent volume at a 3-week interval and were followed up until Day 50 in this interim analysis. RESULTS: In the preclinical studies, S-268019-a was safe and elicited robust immunoglobulin G (IgG) and neutralizing antibody responses in mice. When challenged with SARS-CoV-2, all S-268019-a-treated mice survived and maintained weight until 10 days, whereas all placebo- or adjuvant-treated (without antigen) mice died within 6 days. In the Phase 1/2 trial, although S-268019-a was well tolerated in adult participants, was safe up to Day 50, and elicited robust anti-spike protein IgG antibodies, it did not elicit sufficient neutralizing antibody levels. CONCLUSIONS: The S-268019-a vaccine was not sufficiently immunogenic in Japanese adults despite robust immunogenicity and efficacy in mice. Our results exemplify the innate challenges in translating preclinical data in animals to clinical trials, and highlight the need for continued research to overcome such barriers. (jRCT2051200092).

Immune response and protective efficacy of the SARS-CoV-2 recombinant spike protein vaccine S-268019-b in mice.

Vaccines that efficiently target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent for coronavirus disease (COVID-19), are the best means for controlling viral spread. This study evaluated the efficacy of the COVID-19 vaccine S-268019-b, which comprises the recombinant full-length SARS-CoV-2 spike protein S-910823 (antigen) and A-910823 (adjuvant). In addition to eliciting both Th1-type and Th2-type cellular immune responses, two doses of S-910823 plus A-910823 induced anti-spike protein IgG antibodies and neutralizing antibodies against SARS-CoV-2. In a SARS-CoV-2 challenge test, S-910823 plus A-910823 mitigated SARS-CoV-2 infection-induced weight loss and death and inhibited viral replication in mouse lungs. S-910823 plus A-910823 promoted cytokine and chemokine at the injection site and immune cell accumulation in the draining lymph nodes. This led to the formation of germinal centers and the induction of memory B cells, antibody-secreting cells, and memory T cells. These findings provide fundamental property of S-268019-b, especially importance of A-910823 to elicit humoral and cellular immune responses.

Homologous and heterologous booster vaccinations of S-268019-b, a recombinant S protein-based vaccine with a squalene-based adjuvant, enhance neutralization breadth against SARS-CoV-2 Omicron subvariants in cynomolgus macaques.

SARS-CoV-2 Omicron subvariants such as BA.2.12.1, BA.4 and BA.5 have been spreading rapidly and become dominant worldwide. Here we report the homologous or heterologous booster effects of S-268019-b, a recombinant spike protein vaccine with the squalene-based adjuvant A-910823 in cynomolgus macaques. In macaques which had been primed with S-268019-b or mRNA vaccines, boosting with S-268019-b enhanced neutralizing antibodies (NAb) against ancestral SARS-CoV-2. Since boosting with the antigen without adjuvant did not efficiently restore NAb titers, adjuvant A-910823 was essential for the booster effect. Importantly, boosting with S-268019-b enhanced NAb against all of the Omicron subvariants we tested, including BA.2.12.1, BA.4 and BA.5, in comparison to two vaccine doses. Additionally, expansion of Omicron-specific B cells was confirmed after boosting with S-268019-b. These results indicate that a booster dose of S-268019-b with the adjuvant enhances the neutralization breadth.

Assessing the fitness of a dual-antiviral drug resistant human influenza virus in the ferret model.

Influenza antivirals are important tools in our fight against annual influenza epidemics and future influenza pandemics. Combinations of antivirals may reduce the likelihood of drug resistance and improve clinical outcomes. Previously, two hospitalised immunocompromised influenza patients, who received a combination of a neuraminidase inhibitor and baloxavir marboxil, shed influenza viruses resistant to both drugs. Here-in, the replicative fitness of one of these A(H1N1)pdm09 virus isolates with dual resistance mutations (NA-H275Y and PA-I38T) was similar to wild type virus (WT) in vitro, but reduced in the upper respiratory tracts of challenged ferrets. The dual-mutant virus transmitted well between ferrets in an airborne transmission model, but was outcompeted by the WT when the two viruses were co-administered. These results indicate the dual-mutant virus had a moderate loss of viral fitness compared to the WT virus, suggesting that while person-to-person transmission of the dual-resistant virus may be possible, widespread community transmission is unlikely.

Immunogenicity and protective efficacy of SARS-CoV-2 recombinant S-protein vaccine S-268019-b in cynomolgus monkeys.

The vaccine S-268019-b is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-protein vaccine consisting of full-length recombinant SARS-CoV-2 S-protein (S-910823) as antigen, mixed with the squalene-based adjuvant A-910823. The current study evaluated the immunogenicity of S-268019-b using various doses of S-910823 and its vaccine efficacy against SARS-CoV-2 challenge in cynomolgus monkeys. The different doses of S-910823 combined with A-910823 were intramuscularly administered twice at a 3-week interval. Two weeks after the second dosing, dose-dependent humoral immune responses were observed with neutralizing antibody titers being comparable to that of human convalescent plasma. Pseudoviruses harboring S proteins from Beta and Gamma SARS-CoV-2 variants displayed approximately 3- to 4-fold reduced sensitivity to neutralizing antibodies induced after two vaccine doses compared with that against ancestral viruses, whereas neutralizing antibody titers were reduced >14-fold against the Omicron variant. Cellular immunity was also induced with a relative Th1 polarized response. No adverse clinical signs or weight loss associated with the vaccine were observed, suggesting safety of the vaccine in cynomolgus monkeys. Immunization with 10 µg of S-910823 with A-910823 demonstrated protective efficacy against SARS-CoV-2 challenge according to genomic and subgenomic viral RNA transcript levels in nasopharyngeal, throat, and rectal swab specimens. Pathological analysis revealed no detectable vaccine-dependent enhancement of disease in the lungs of challenged vaccinated monkeys. The current findings provide fundamental information regarding vaccine doses for human trials and support the development of S-268019-b as a safe and effective vaccine for controlling the current pandemic, as well as general protection against SARS-CoV-2 moving forward.

Immunogenicity and safety of booster dose of S-268019-b or BNT162b2 in Japanese participants: An interim report of phase 2/3, randomized, observer-blinded, noninferiority study.

In this randomized, observer-blinded, phase 2/3 study, S-268019-b (n = 101), a recombinant spike protein vaccine, was analyzed for noninferiority versus BNT162b2 (n = 103), when given as a booster ≥6 months after 2-dose BNT162b2 regimen in Japanese adults without prior SARS-CoV-2 infection. Interim results showed noninferiority of S-268019-b versus BNT162b2 in co-primary endpoints for neutralizing antibodies on day 29: geometric mean titer (GMT) (124.97 versus 109.70; adjusted-GMT ratio [95% CI], 1.14 [0.94-1.39]; noninferiority P-value, <0.0001) and seroresponse rate (both 100%; noninferiority P-value, 0.0004). Both vaccines elicited anti-spike-protein immunoglobulin G antibodies, and produced T-cell response (n = 29/group) and neutralizing antibodies against Delta and Omicron pseudovirus and live virus variants (n = 24/group) in subgroups. Most participants reported low-grade reactogenicity on days 1-2, the most frequent being fatigue, fever, myalgia, and injection-site pain. No serious adverse events were reported. In conclusion, S-268019-b was safe and showed robust immunogenicity as a booster, supporting its use as COVID-19 booster vaccine.

Phase 1/2 clinical trial of COVID-19 vaccine in Japanese participants: A report of interim findings.

We initiated a randomized, placebo-controlled, phase 1/2 trial to evaluate the safety and immunogenicity of the S-268019-b recombinant protein vaccine, scheduled as 2 intramuscular injections given 21 days apart, in 60 randomized healthy Japanese adults. We evaluated 2 regimens of the S-910823 antigen (5 µg [n = 24] and 10 µg [n = 24]) with an oil-in-water emulsion formulation and compared against placebo (n = 12). Reactogenicity was mild in most participants. No serious adverse events were noted. For both regimens, vaccination resulted in robust IgG and neutralizing antibody production at days 36 and 50 and predominant T-helper 1-mediated immune reaction, as evident through antigen-specific polyfunctional CD4+ T-cell responses with IFN-γ, IL-2, and IL-4 production on spike protein peptides stimulation. Based on the interim analysis, the S-268019-b vaccine is safe, produces neutralizing antibodies titer comparable with that in convalescent serum from COVID-19-recovered patients. However, further evaluation of the vaccine in a large clinical trial is warranted.

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model.

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.

Comprehensive assessment of amino acid substitutions in the trimeric RNA polymerase complex of influenza A virus detected in clinical trials of baloxavir marboxil.

BACKGROUND: Baloxavir marboxil (BXM) is an approved drug that selectively targets cap-dependent endonuclease on PA subunit in the RNA polymerase complex of influenza A and B viruses. Amino acid substitutions at position 38 in the PA subunit were identified as a major pathway for reduced susceptibility to baloxavir acid (BXA), the active form of BXM. Additionally, substitutions found at positions E23, A37, and E199 in the PA subunit impact BXA susceptibility by less than 10-fold. METHODS: We comprehensively evaluated the impact of novel amino acid substitutions identified in PA, PB1, and PB2 subunits in BXM clinical trials and influenza sequence databases by means of drug susceptibility and replicative capacity. RESULTS: PA/I38N in A(H1N1)pdm09 and PA/I38R in A(H3N2) were newly identified as treatment-emergent substitutions in the CAPSTONE-2 study. The I38N substitution conferred reduced susceptibility by 24-fold, whereas replicative capacity of the I38N-substituted virus was impaired compared with the wild-type. The I38R-substituted virus was not viable in cell culture. All other mutations assessed in this extensive study did not significantly affect BXA susceptibility (< 2.4-fold change). CONCLUSION: These results provide additional information on the impact of amino acid substitutions in the trimeric viral polymerase complex to BXA susceptibility and will further support influenza surveillance.

Carryover effects of baloxavir acid in human nasopharyngeal/pharyngeal swabs on infectious titer testing of influenza virus.

Baloxavir marboxil (BXM) demonstrated a rapid and profound decline in infectious viral titer 1 day after BXM administration. Rapid reduction in virus titer is a characteristic of BXM. There may be a possibility that drug carryover effects have impacts on the observed antiviral effects due to the poor correlation that was observed between viral titer reduction and alleviation of influenza symptoms. Here, we report possible carryover effects of baloxavir acid (BXA), an active form of BXM, on infectious titer testing. Our findings indicate that there is little impact of BXA carryover on the infectious titer testing.

Treatment-Emergent Influenza Variant Viruses With Reduced Baloxavir Susceptibility: Impact on Clinical and Virologic Outcomes in Uncomplicated Influenza.

BACKGROUND: Single-dose baloxavir rapidly reduces influenza virus titers and symptoms in patients with uncomplicated influenza, but viruses with reduced in vitro susceptibility due to amino acid substitutions at position 38 of polymerase acidic protein (PA/I38X) sometimes emerge. METHODS: We evaluated the kinetics, risk factors, and effects on clinical and virologic outcomes of emergence of PA/I38X-substituted viruses. RESULTS: Viruses containing PA/I38X substitutions were identified 3-9 days after baloxavir treatment in 9.7% (36/370) of patients, of whom 85.3% had transient virus titer rises. Median time to sustained cessation of infectious virus detection was 192, 48, and 96 hours in the baloxavir recipients with PA/I38X-substituted viruses, without PA/I38X-substituted viruses, and placebo recipients, respectively. The corresponding median times to alleviation of symptoms were 63.1, 51.0, and 80.2 hours, respectively. After day 5, symptom increases occurred in 11.5%, 8.0%, and 13.0%, respectively, and in 8.9% of oseltamivir recipients. Variant virus emergence was associated with lower baseline neutralizing antibody titers. CONCLUSIONS: The emergence of viruses with PA/I38X substitutions following baloxavir treatment was associated with transient rises in infectious virus titers, prolongation of virus detectability, initial delay in symptom alleviation, and uncommonly with symptom rebound. The potential transmissibility of PA/I38X-substituted viruses requires careful study. CLINICAL TRIAL REGISTRATION: NCT02954354.

Baloxavir Marboxil in Japanese Pediatric Patients With Influenza: Safety and Clinical and Virologic Outcomes.

BACKGROUND: We assessed the safety and effectiveness of baloxavir marboxil administration in Japanese children with influenza. METHODS: This open-label study administered 1 weight-adjusted dose of baloxavir to 107 children aged 1-11 years with laboratory-confirmed, febrile influenza virus infection of ≤48 hours duration. RESULTS: Adverse events (AEs) were reported in 34.6% of patients, most commonly vomiting (7.5%); no serious AEs or AEs causing discontinuation occurred. The median time to alleviation of influenza illness was 44.6 hours (95% confidence interval, 38.9-62.5 hours), to resolution of fever was 21.4 hours, and to sustained cessation of infectious viral shedding was 24.0 hours. However, viruses with amino acid substitutions in the viral polymerase acidic protein at position I38 (PA/I38T/M) emerged in 18 of 77 (23.4%) patients. Emergence was associated with longer infectious virus detectability (median time, 180.0 hours) and time to illness alleviation (median, 79.6 vs 42.8 hours in patients without PA/I38T/M-substituted viruses). Among patients with PA/I38T/M-substituted virus emergence, those with baseline hemagglutinin inhibition (HAI) antibody titer <40 experienced delay in time to illness alleviation (median, 85.4 vs 56.0 hours in patients with higher baseline HAI antibody titer). CONCLUSIONS: A single, oral dose of baloxavir marboxil was well tolerated and rapidly reduced viral titers, but the common emergence of PA/I38T/M-substituted viruses warrants consideration of alternative dosing regimens in young children. CLINICAL TRIALS REGISTRATION: Japan Pharmaceutical Information Center Clinical Trials Information (Japic CTI-163417).

In vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase PA subunit.

Cap-dependent endonuclease (CEN) resides in the PA subunit of the influenza virus and mediates the critical "cap-snatching" step of viral RNA transcription, which is considered to be a promising anti-influenza target. Here, we describe in vitro characterization of a novel CEN inhibitor, baloxavir acid (BXA), the active form of baloxavir marboxil (BXM). BXA inhibits viral RNA transcription via selective inhibition of CEN activity in enzymatic assays, and inhibits viral replication in infected cells without cytotoxicity in cytopathic effect assays. The antiviral activity of BXA is also confirmed in yield reduction assays with seasonal type A and B viruses, including neuraminidase inhibitor-resistant strains. Furthermore, BXA shows broad potency against various subtypes of influenza A viruses (H1N2, H5N1, H5N2, H5N6, H7N9 and H9N2). Additionally, serial passages of the viruses in the presence of BXA result in isolation of PA/I38T variants with reduced BXA susceptibility. Phenotypic and genotypic analyses with reverse genetics demonstrate the mechanism of BXA action via CEN inhibition in infected cells. These results reveal the in vitro characteristics of BXA and support clinical use of BXM to treat influenza.

Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil.

Baloxavir acid (BXA), derived from the prodrug baloxavir marboxil (BXM), potently and selectively inhibits the cap-dependent endonuclease within the polymerase PA subunit of influenza A and B viruses. In clinical trials, single doses of BXM profoundly decrease viral titers as well as alleviating influenza symptoms. Here, we characterize the impact on BXA susceptibility and replicative capacity of variant viruses detected in the post-treatment monitoring of the clinical studies. We find that the PA I38T substitution is a major pathway for reduced susceptibility to BXA, with 30- to 50-fold and 7-fold EC50 changes in A and B viruses, respectively. The viruses harboring the I38T substitution show severely impaired replicative fitness in cells, and correspondingly reduced endonuclease activity in vitro. Co-crystal structures of wild-type and I38T influenza A and B endonucleases bound to BXA show that the mutation reduces van der Waals contacts with the inhibitor. A reduced affinity to the I38T mutant is supported by the lower stability of the BXA-bound endonuclease. These mechanistic insights provide markers for future surveillance of treated populations.

Suppression of RIP3-dependent necroptosis by human cytomegalovirus.

Necroptosis is an alternate programmed cell death pathway that is unleashed by caspase-8 compromise and mediated by receptor-interacting protein kinase 3 (RIP3). Murine cytomegalovirus (CMV) and herpes simplex virus (HSV) encode caspase-8 inhibitors that prevent apoptosis together with competitors of RIP homotypic interaction motif (RHIM)-dependent signal transduction to interrupt the necroptosis. Here, we show that pro-necrotic murine CMV M45 mutant virus drives virus-induced necroptosis during nonproductive infection of RIP3-expressing human fibroblasts, whereas WT virus does not. Thus, M45-encoded RHIM competitor, viral inhibitor of RIP activation, sustains viability of human cells like it is known to function in infected mouse cells. Importantly, human CMV is shown to block necroptosis induced by either TNF or M45 mutant murine CMV in RIP3-expressing human cells. Human CMV blocks TNF-induced necroptosis after RIP3 activation and phosphorylation of the mixed lineage kinase domain-like (MLKL) pseudokinase. An early, IE1-regulated viral gene product acts on a necroptosis step that follows MLKL phosphorylation prior to membrane leakage. This suppression strategy is distinct from RHIM signaling competition by murine CMV or HSV and interrupts an execution process that has not yet been fully elaborated.

Herpes simplex virus suppresses necroptosis in human cells.

Herpes simplex virus (HSV)-1 and HSV-2 are significant human pathogens causing recurrent disease. During infection, HSV modulates cell death pathways using the large subunit (R1) of ribonucleotide reductase (RR) to suppress apoptosis by binding to and blocking caspase-8. Here, we demonstrate that HSV-1 and HSV-2 R1 proteins (ICP6 and ICP10, respectively) also prevent necroptosis in human cells by inhibiting the interaction between receptor-interacting protein kinase 1 (RIP1) and RIP3, a key step in tumor necrosis factor (TNF)-induced necroptosis. We show that suppression of this cell death pathway requires an N-terminal RIP homotypic interaction motif (RHIM) within R1, acting in concert with the caspase-8-binding domain, which unleashes necroptosis independent of RHIM function. Thus, necroptosis is a human host defense pathway against two important viral pathogens that naturally subvert multiple death pathways via a single evolutionarily conserved gene product.