Sixty Years of Discovery.

Each of us is a story. Mine is a story of doing science for 60 years, and I am honored to be asked to tell it. Even though this autobiography was written for the Annual Review of Immunology, I have chosen to describe my whole career in science because the segment that was immunology is so intertwined with all else I was doing. This article is an elongation and modification of a talk I gave at my 80th birthday celebration at Caltech on March 23, 2018.

T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis.

Although T cells can exert potent anti-tumor immunity, a subset of T helper (Th) cells producing interleukin-22 (IL-22) in breast and lung tumors is linked to dismal patient outcome. Here, we examined the mechanisms whereby these T cells contribute to disease. In murine models of lung and breast cancer, constitutional and T cell-specific deletion of Il22 reduced metastases without affecting primary tumor growth. Deletion of the IL-22 receptor on cancer cells decreases metastasis to a degree similar to that seen in IL-22-deficient mice. IL-22 induced high expression of CD155, which bound to the activating receptor CD226 on NK cells. Excessive activation led to decreased amounts of CD226 and functionally impaired NK cells, which elevated the metastatic burden. IL-22 signaling was also associated with CD155 expression in human datasets and with poor patient outcomes. Taken together, our findings reveal an immunosuppressive circuit activated by T cell-derived IL-22 that promotes lung metastasis.

Immunity to enteric viruses.

Pathogenic enteric viruses are a major cause of morbidity and mortality, particularly among children in developing countries. The host response to enteric viruses occurs primarily within the mucosa, where the intestinal immune system must balance protection against pathogens with tissue protection and tolerance to harmless commensal bacteria and food. Here, we summarize current knowledge in natural immunity to enteric viruses, highlighting specialized features of the intestinal immune system. We further discuss how knowledge of intestinal anti-viral mechanisms can be translated into vaccine development with particular focus on immunization in the oral route. Research reveals that the intestine is a complex interface between enteric viruses and the host where environmental factors influence susceptibility and immunity to infection, while viral infections can have lasting implications for host health. A deeper mechanistic understanding of enteric anti-viral immunity with this broader context can ultimately lead to better vaccines for existing and emerging viruses.

Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

High-resolution mapping reveals the mechanism and contribution of genome insertions and deletions to RNA virus evolution.

RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions (indels) in viral genomes are major contributors to both deleterious mutational load and evolutionary novelty, but remain understudied. To characterize the mechanistic details of their formation and evolutionary dynamics during infection, we developed a hybrid experimental-bioinformatic approach. This approach, called MultiMatch, extracts insertions and deletions from ultradeep sequencing experiments, including those occurring at extremely low frequencies, allowing us to map their genomic distribution and quantify the rates at which they occur. Mapping indel mutations in adapting poliovirus and dengue virus populations, we determine the rates of indel generation and identify mechanistic and functional constraints shaping indel diversity. Using poliovirus RdRp variants of distinct fidelity and genome recombination rates, we demonstrate tradeoffs between fidelity and Indel generation. Additionally, we show that maintaining translation frame and viral RNA structures constrain the Indel landscape and that, due to these significant fitness effects, Indels exert a significant deleterious load on adapting viral populations. Conversely, we uncover positively selected Indels that modulate RNA structure, generate protein variants, and produce defective interfering genomes in viral populations. Together, our analyses establish the kinetic and mechanistic tradeoffs between misincorporation, recombination, and Indel rates and reveal functional principles defining the central role of Indels in virus evolution, emergence, and the regulation of viral infection.

Two-Year Duration of Immunity of Inactivated Poliovirus Vaccine: A Follow-up Study in Pakistan in 2020.

This was a follow-up study conducted in 2020 assessing changes in levels of type 2 poliovirus-neutralizing antibodies 2 years postimmunization in children who received inactivated poliovirus vaccine (IPV) in Karachi, Pakistan. Unexpectedly, the findings revealed an increase in seroprevalence of type 2 antibodies from 73.1% to 81.6% 1 year and 2 years after IPV, respectively. The increase in type 2 immunity could result from the intensive transmission of circulating vaccine-derived poliovirus type 2 (cVDPV2) in Karachi during the second year of IPV administration. This study suggests that the cVDPV2 outbreak detected in Pakistan infected large proportions of children in Karachi. Clinical Trials Registration . NCT03286803.

Impact of Supplementary Immunization Activities using Novel Oral Polio Vaccine Type 2 during a Large outbreak of Circulating Vaccine-Derived Poliovirus in Nigeria.

BACKGROUND: Novel oral poliovirus vaccine (OPV) type 2 (nOPV2) has been made available for outbreak response under an emergency use listing authorization based on supportive clinical trial data. Since 2021 more than 350 million doses of nOPV2 were used for control of a large outbreak of circulating vaccine-derived poliovirus type 2 (cVDPV2) in Nigeria. METHODS: Using a bayesian time-series susceptible-infectious-recovered model, we evaluate the field effectiveness of nOPV2 immunization campaigns in Nigeria compared with campaigns using monovalent OPV type 2 (mOPV2). RESULTS: We found that both nOPV2 and mOPV2 campaigns were highly effective in reducing transmission of cVDPV2, on average reducing the susceptible population by 42% (95% confidence interval, 28-54%) and 38% (20-51%) per campaign, respectively, which were indistinguishable from each other in this analysis (relative effect, 1.1 [.7-1.9]). Impact was found to vary across areas and between immunization campaigns. CONCLUSIONS: These results are consistent with the comparable individual immunogenicity of nOPV2 and mOPV2 found in clinical trials but also suggest that outbreak response campaigns may have small impacts in some areas requiring more campaigns than are suggested in current outbreak response procedures.

Progress in Access and Oral Polio Vaccine Coverage Among Children Aged <5 Years in Polio Campaigns After the Political Change in Afghanistan.

BACKGROUND: Warfare has long impeded vaccination programs in polio-endemic Afghanistan. We aimed to describe progress in access to children under 5, oral polio vaccine (OPV) coverage among children under 5 in nationwide polio campaigns, and polio surveillance performance indicators after the Islamic Republic of Afghanistan collapsed to Taliban forces in August 2021. METHODS: Trends in the number of wild poliovirus type 1 (WPV1) and circulating vaccine-derived poliovirus type 2 (cVDPV2) cases and surveillance indicators from 2015 to 2023, and trends in the OPV coverage in the November 2020-June 2022 polio campaigns, were described. RESULTS: From 2015 to mid-July 2020, 74 of 126 (58.7%) WPV1 cases were reported from inaccessible areas. In November 2020, 34.1% of target children under 5 were inaccessible; in November 2021 (the first postchange polio campaign), all were accessible. From November 2020, under-5 OPV coverage of 69.9% rose steadily to 99.9% in the May 2022 campaign. The number of cVDPV cases fell from 308 (2020) to zero (2022). June 2022's house-to-house OPV coverage was 34.2% higher than non-house-to-house modalities. Nonpolio acute flaccid paralysis and stool adequacy rates rose from 18.5/100 000 and 92.6% in 2020 to 24.3/100 000 and 94.4% in 2022, respectively. CONCLUSIONS: Children's inaccessibility no longer vitiates polio eradication; polio surveillance systems are less likely to miss any poliovirus circulation.

Nasal and pharyngeal mucosal immunity to poliovirus in children following routine immunization with inactivated polio vaccine (IPV) in the United States of America.

BACKGROUND: Although polioviruses (PVs) replicate in lymphoid tissue of both the pharynx and ileum, research on polio vaccine-induced mucosal immunity has predominantly focused on intestinal neutralizing and binding antibody levels measured in stool. METHODS: To investigate the extent to which routine immunization with intramuscularly injected inactivated polio vaccine (IPV) may induce nasal and pharyngeal mucosal immunity, we measured PV type-specific neutralization and immunoglobulin (Ig) G, IgA, and IgM levels in nasal secretions, adenoid cell supernatants, and sera collected from 12 children, aged 2 to 5 years, undergoing planned adenoidectomies. All participants were routinely immunized with IPV and had no known contact with live PVs. RESULTS: PV-specific mucosal neutralization was detected in nasal and adenoid samples, mostly from children who had previously received four IPV doses. Across the three PV serotypes, both nasal (Spearman's rho ≥ 0.87, p≤0.0003 for all) and adenoid (Spearman's rho ≥0.57, p≤0.05 for all) neutralization titers correlated with serum neutralization titers. In this small study sample, there was insufficient evidence to determine which Ig isotype(s) was correlated with neutralization. CONCLUSIONS: Our findings provide policy-relevant evidence that routine immunization with IPV may induce nasal and pharyngeal mucosal immunity. The observed correlations of nasal and pharyngeal mucosal neutralization with serum neutralization contrast with previous observations of distinct intestinal and serum responses to PV vaccines. Further research is warranted to determine which antibody isotype(s) correlate with polio vaccine-induced nasal and pharyngeal mucosal neutralizing activity and to understand the differences from intestinal mucosal immunity.

Mucosal immunity to poliovirus in children 0-15 years of age: A community-based study in Karachi, Pakistan in 2019.

This study assesses poliovirus type 1 (PV1) immunity in children to inform the contribution of mucosal immunity in and preventing poliovirus circulation. A community-based study was conducted in peri-urban Karachi, Pakistan. Randomly selected children (0-15 years) received oral poliovirus vaccine (OPV) challenge dose. Blood and stool samples were collected at several time points and evaluated for polio-neutralizing antibodies and serotype-specific poliovirus, respectively. 81/589 (14%) children excreted PV1 7 days post-OPV-challenge; 70/81 (86%) were seropositive at baseline. 12/610 (2%) were asymptomatic Wild Poliovirus Type 1 (WPV1) excretors. Most poliovirus excretors had humoral immunity, suggesting mucosal immunity in these children likely waned or never developed. Without mucosal immunity, they are susceptible to poliovirus infection, shedding, and transmission. Asymptomatic WPV1 excretion suggests undetected poliovirus circulation within the community.

Novel Oral Poliovirus Vaccine 2 Safety Evaluation during Nationwide Supplemental Immunization Activity, Uganda, 2022.

Given its enhanced genetic stability, novel oral poliovirus vaccine type 2 was deployed for type 2 poliovirus outbreak responses under World Health Organization Emergency Use Listing. We evaluated the safety profile of this vaccine. No safety signals were identified using a multipronged approach of passive and active surveillance.

ADAR-mediated RNA editing regulates PVR immune checkpoint in colorectal cancer.

Recent studies have revealed that tumor immunotherapy resistance is influenced by ADAR-mediated RNA editing, but its targets remain unelucidated. Our current study identified the poliovirus receptor (PVR) oncogene, which encodes an immune checkpoint in colorectal cancer (CRC), as a potential target for RNA editing. We performed transcriptome sequencing analysis and experimental validation in two Chinese CRC cohorts. PVR and ADAR expressions significantly increased in CRC tumors and showed positive correlations in both cohorts, coupled with upregulated PVR RNA editing in CRC tumors. Manipulation of ADAR expression by over-expression or knockdown substantially changed PVR expression and RNA editing in HTC116 CRC cells. Luciferase reporter and actinomycin D assays further revealed that RNA editing in PVR 3'-UTR could upregulate PVR RNA expression, probably by increasing the RNA stability. By increasing PVR expression, ADAR-mediate RNA editing might contribute to tumor- and immune-related gene functions and pathways in CRC. Moreover, a signature combining PVR RNA editing and expression showed promising predictive performance in CRC diagnosis in both Chinese CRC cohorts. Our findings thus highlight the importance of ADAR-mediated RNA editing in PVR up-regulation in CRC tumors and provide new insight into the application of PVR RNA editing as a novel diagnostic biomarker for CRC.

Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds.

IMPORTANCE: Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.

Poliomyelitis and COVID-19: a repeated history.

The measurement of the enterovirus and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage water is relevant in the early detection of the introduction or disappearance of these viruses in the ecosystem. We evaluated the co-circulation of the enteroviruses and SARS-CoV-2 in 81 sewage water samples collected between September 2021 and April 2023 from different regions of north and southeast Romania, at the border with Ukraine. We used, for the molecular detection of the pathogens, the multiplex real-time polymerase chain reaction (PCR) assay produced for respiratory samples and the Respiratory 2.1 Plus panel Biofire Film array. The isolation of enteroviruses was performed on cell culture lines, in accordance with the World Health Organization (WHO) recommendations. By molecular investigations, we detected the SARS-CoV-2 in 22 (27%) samples, and the human rhinovirus/enterovirus in 64 (79%) samples. By isolation on cell culture lines, 27 samples (33,33%) were positive for non-polio enteroviruses, and no poliovirus strains were isolated, proving the maintenance of the polio-free status in Romania. In an emergency situation, the molecular detection of the pathogens in sewage water using a PCR system integrating sample preparation, amplification, detection, and analysis in 1 h could be implemented.

Effective partnership and in-country resource mobilization in Sudan for cVDPV2 outbreak response amid multiple emergencies in 2020-2021.

BACKGROUND: During 2020 and immediately prior to the COVID-19 pandemic, Sudan was experiencing multiple emergencies including violence, seasonal flooding, and vector-borne disease outbreaks. After more than ten years since its last case of wild poliovirus, Sudan declared a circulating vaccine-derived poliovirus type 2 (cVDPV2) outbreak on 9 August 2020. METHODS: cVDPV2 outbreak response data and programme documents of the Federal Ministry of Health and WHO were reviewed. Surveillance data was verified through WHO-recommended procedures for detecting and characterizing polioviruses from stool and sewage samples collected from acute flaccid paralysis (AFP) cases and the environment. RESULTS: This outbreak in Sudan led to a total of 58 confirmed cases of cVDPV2 from 15 of the 18 states. Two nationwide vaccination campaigns were held to increase immunity of children under-five against poliovirus type 2. Funding challenges were overcome by intense additional resource mobilization from in-country sources. The funding gap was bridged from domestic resources (49%) sourced through GPEI partners, and in-country humanitarian funding mechanisms. CONCLUSIONS: During an outbreak response and challenge of funding shortfall, mobilizing in-country resources is possible through coordinated approaches, regular communication with partners, disaggregation of needs, and matching in-kind and financial support to fill gaps. A cVDPV2 outbreak requires a fast, resourced, and quality response to stop virus circulation.

Coordinated global cessation of oral poliovirus vaccine use: Options and potential consequences.

Due to the very low, but nonzero, paralysis risks associated with the use of oral poliovirus vaccine (OPV), eradicating poliomyelitis requires ending all OPV use globally. The Global Polio Eradication Initiative (GPEI) coordinated cessation of Sabin type 2 OPV (OPV2 cessation) in 2016, except for emergency outbreak response. However, as of early 2023, plans for cessation of bivalent OPV (bOPV, containing types 1 and 3 OPV) remain undefined, and OPV2 use for outbreak response continues due to ongoing transmission of type 2 polioviruses and reported type 2 cases. Recent development and use of a genetically stabilized novel type 2 OPV (nOPV2) leads to additional potential vaccine options and increasing complexity in strategies for the polio endgame. Prior applications of integrated global risk, economic, and poliovirus transmission modeling consistent with GPEI strategic plans that preceded OPV2 cessation explored OPV cessation dynamics and the evaluation of options to support globally coordinated risk management efforts. The 2022-2026 GPEI strategic plan highlighted the need for early bOPV cessation planning. We review the published modeling and explore bOPV cessation immunization options as of 2022, assuming that the GPEI partners will not support restart of the use of any OPV type in routine immunization after a globally coordinated cessation of such use. We model the potential consequences of globally coordinating bOPV cessation in 2027, as anticipated in the 2022-2026 GPEI strategic plan. We do not find any options for bOPV cessation likely to succeed without a strategy of bOPV intensification to increase population immunity prior to cessation.

Worst-case scenarios: Modeling uncontrolled type 2 polio transmission.

In May 2016, the Global Polio Eradication Initiative (GPEI) coordinated the cessation of all use of type 2 oral poliovirus vaccine (OPV2), except for emergency outbreak response. Since then, paralytic polio cases caused by type 2 vaccine-derived polioviruses now exceed 3,000 cases reported by 39 countries. In 2022 (as of April 25, 2023), 20 countries reported detection of cases and nine other countries reported environmental surveillance detection, but no reported cases. Recent development of a genetically modified novel type 2 OPV (nOPV2) may help curb the generation of neurovirulent vaccine-derived strains; its use since 2021 under Emergency Use Listing is limited to outbreak response activities. Prior modeling studies showed that the expected trajectory for global type 2 viruses does not appear headed toward eradication, even with the best possible properties of nOPV2 assuming current outbreak response performance. Continued persistence of type 2 poliovirus transmission exposes the world to the risks of potentially high-consequence events such as the importation of virus into high-transmission areas of India or Bangladesh. Building on prior polio endgame modeling and assuming current national and GPEI outbreak response performance, we show no probability of successfully eradicating type 2 polioviruses in the near term regardless of vaccine choice. We also demonstrate the possible worst-case scenarios could result in rapid expansion of paralytic cases and preclude the goal of permanently ending all cases of poliomyelitis in the foreseeable future. Avoiding such catastrophic scenarios will depend on the development of strategies that raise population immunity to type 2 polioviruses.

Observational analysis of the immunogenicity and safety of various types of spinal muscular atrophy vaccines.

BACKGROUND: This study aimed to evaluate the immunogenicity and safety of different types of poliovirus vaccines. METHODS: A randomized, blinded, single-center, parallel-controlled design was employed, and 360 infants aged ≥ 2 months were selected as study subjects. They were randomly assigned to bOPV group (oral Sabin vaccine) and sIPV group (Sabin strain inactivated polio vaccine), with 180 infants in each group. Adverse reaction events in the vaccinated subjects were recorded. The micro-neutralization test using cell culture was conducted to determine the geometric mean titer (GMT) of neutralizing antibodies against poliovirus types I, II, and III in different groups, and the seroconversion rates were calculated. RESULTS: Both groups exhibited a 100% seropositivity rate after booster immunization. The titers of neutralizing antibodies for the three types were predominantly distributed within the range of 1:128 to 1:512. The fold increase of type I antibodies differed markedly between the two groups (P < 0.05). Moreover, the fold increase of type II and type III antibodies for poliovirus differed slightly between the two groups (P > 0.05). The fourfold increase rate in sIPV group was drastically superior to that in bOPV group (P < 0.05). When comparing the post-immunization GMT levels of type I antibodies in individuals who completed the full course of spinal muscular atrophy vaccination, bOPV group showed greatly inferior levels to sIPV group (P < 0.05). For type II and type III antibodies, individuals in bOPV group demonstrated drastically superior post-immunization GMT levels to those in sIPV group (P < 0.05). The incidence of adverse reactions between the bOPV and sIPV groups differed slightly (P > 0.05). CONCLUSION: These findings indicated that both the oral vaccine and inactivated vaccine had good safety and immunogenicity in infants aged ≥ 2 months. The sIPV group generated higher levels of neutralizing antibodies in serum, particularly evident in the post-immunization GMT levels for types II and III.

Response to Vaccine-Derived Polioviruses Detected through Environmental Surveillance, Guatemala, 2019.

Guatemala implemented wastewater-based poliovirus surveillance in 2018, and three genetically unrelated vaccine-derived polioviruses (VDPVs) were detected in 2019. The Ministry of Health (MoH) response included event investigation through institutional and community retrospective case searches for acute flaccid paralysis (AFP) during 2018-2020 and a bivalent oral polio/measles, mumps, and rubella vaccination campaign in September 2019. This response was reviewed by an international expert team in July 2021. During the campaign, 93% of children 6 months <7 years of age received a polio-containing vaccine dose. No AFP cases were detected in the community search; institutional retrospective searches found 37% of unreported AFP cases in 2018‒2020. No additional VDPV was isolated from wastewater. No evidence of circulating VDPV was found; the 3 isolated VDPVs were classified as ambiguous VDPVs by the international team of experts. These detections highlight risk for poliomyelitis reemergence in countries with low polio vaccine coverage.

MicroRNA-326 negatively regulates CD155 expression in lung adenocarcinoma.

Treatment with immune checkpoint inhibitors induces a durable response in some patients with non-small-cell lung cancer, but eventually gives rise to drug resistance. Upregulation of CD155 expression is implicated as one mechanism of resistance to programmed death receptor-1 (PD-1)/PD-1 ligand (PD-L1) inhibitors, and it is therefore important to characterize the mechanisms underlying regulation of CD155 expression in tumor cells. The aim of this study was to identify microRNAs (miRNAs) that might regulate CD155 expression at the posttranscriptional level in lung cancer. Comprehensive miRNA screening with target prediction programs and a dual-luciferase reporter assay identified miR-346, miR-328-3p, miR-326, and miR-330-5p as miRNAs that bind to the 3'-UTR of CD155 mRNA. Forced expression of these miRNAs suppressed CD155 expression in lung cancer cell lines. Immunohistochemical staining of CD155 in tissue specimens from 57 patients with lung adenocarcinoma revealed the median tumor proportion score for CD155 to be 68%. The abundance of miR-326 in these specimens with a low level of CD155 expression was significantly greater than in specimens with a high level (p < 0.005). Our results thus suggest that miR-326 negatively regulates CD155 expression in lung adenocarcinoma and might therefore play a role in the development of resistance to PD-1/PD-L1 inhibitors.