Abstract Background T cells are associated with viral clearance and improved outcomes in COVID-19, but also with immunopathology. Defining T cell diversity in response to SARS-CoV-2 infection enables implementation of T cells in prevention and treatment strategies. T cells recognize peptide sequences from pathogens upon presentation in major histocompatibility complexes (MHC). Human MHC molecule diversity results in a wide array of peptide recognition by T cells making viral escape difficult, but also challenging to measure. Methods Using the ImmunoSEQ Assay to sequence complementarity-determining regions in the β chains (CDR3β) of the T cell receptor (TCR) from peripheral blood collected from participants in the EPICC COVID-19 cohort study, we quantified and characterized patterns of TCR usage. Multiple bioinformatic pipelines were compared for analysis. Results Our study evaluated TCR repertoires from 39 participants who had mild (20 outpatient) versus severe (19 hospitalized) acute COVID-19, and either experienced unvaccinated primary infection (n = 15) or vaccine breakthrough infection (n = 24) (Table 1). From these four clinical groups, we used the ImmuneCODE database, VDJdatabase and healthy controls to identify CD8 TCR sequences and amino acid motifs in the CDR3β region specific for the SARS-CoV-2 proteome. We were surprised to find that vaccine breakthrough and primary infection participants exhibited similar frequencies of SARS-CoV-2-specific TCR sequences over 14-21 days post-symptom onset. Increased use of common TCR clones occurred in participants with older age and comorbidities, which correlated with more severe disease (Figure 1). Strikingly, an increased frequency of non-SARS-CoV-2 TCRs was significantly associated with severe disease in unvaccinated (p = 0.006) and vaccinated participants (p = 0.045). Conclusion In conclusion, our study highlights the opportunities and challenges of studying the complexity of T cell diversity and the utility of combining databases and bioinformatics for identification of patterns of TCR recognition. Interestingly, participants who were hospitalized were more likely to have circulating T cells non-specific for SARS-CoV-2 suggesting that vaccines that increase T cell specificity may reduce disease severity. Disclosures Simon Pollett, MBBS, AstraZeneca: The IDCRP and HJF were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial as part of US Govt COVID Response

Introduction:T cells influence COVID-19 severity and establish long-lasting immune memory in response to vaccination and infection. The diversity of the T cell repertoire, and complexity of T cell epitope recognition, make it challenging to define protective epitope-specific T cells. In this study, we created a highly specific TCR meta-database to identify T cell epitopes from the nearly complete SARS-CoV-2 proteome and determine whether vaccination with mRNA vaccines influenced the TCR repertoire. Methods:Using this meta-database, we analyzed immunosequencing data of genomic DNA to define the variable region of T cell receptor (TCR) b chain (TCRB) sequences among participants in a longitudinal COVID-19 cohort study. The TCR repertoire was compared between participants who were vaccinated or unvaccinated against SARS-CoV-2 and stratified by disease severity. TCR diversity was measured using clonality, an index defined as the inverted normalized Shannon entropy. Results:Highly clonal TCR repertoires correlated with age and comorbidities. Using our meta-database approach, we found that vaccinated participants hospitalized with infection had the most restricted SARS-CoV-2-specific CD8 TCR repertoire. However, TCRB with predicted specificity to non-spike SARS-CoV-2 proteins dominated the response, even in vaccinated participants. We identified a peptide sequence in the ORF10 accessory protein that was more frequently recognized in study participants with mild disease. Conversely, CD8 T cell recognition of a peptide sequence in ORF1ab more closely correlated with severe disease. Discussion:Overarchingly, TCR repertoire analysis revealed that CD8 T cells responding to SARS-CoV-2 broadly recognize epitopes across the SARS-CoV-2 proteome, and provided opportunities to identify epitopes associated with disease.

Ionizable lipid nanoparticles (LNP) that have enabled the success of messenger RNA (mRNA) vaccines have been shown to be immunostimulatory in the absence of mRNA. However, the mechanisms through which they activate innate immune cells is incompletely understood. Using a monocyte cell line, we compared the ability of three LNP formulations to activate transcription factors Nuclear Factor-kappa B (NF-κB) and Interferon Regulatory Factor (IRF). Comparison of signaling in knockout cell lines illustrated a role for Toll-like receptor (TLR) 4 in initiation of this signaling cascade and the contribution of the ionizable lipid component. Activation induced by empty LNPs was similar to that induced by LNPs containing mRNA, indicating that LNPs may provide the majority of innate stimulation for the mRNA vaccine platform. Our findings demonstrate that ionizable lipids within LNPs signal through TLR4 to activate NF-κB and IRF, identifying a mechanism for innate activation that can be optimized for adjuvant design.

Frequencies and phenotypes of immune cells differ between neonates and adults in association with age-specific immune responses. Lymph nodes (LN) are critical tissue sites to quantify and define these differences. Advances in flow cytometry have enabled more multifaceted measurements of complex immune responses. Tissue processing can affect the immune cells under investigation that influence key findings. To understand the impact on immune cells in the LN after processing for single-cell suspension, we compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I treatment, and mechanical dissociation with density gradient separation. We analyzed cell yields, viability, phenotypic and maturation markers of immune cells from the lung-draining LN of neonatal and adult mice two days after intranasal respiratory syncytial virus (RSV) infection. While viability was consistent across age groups, the protocols influenced the yield of subsets defined by important phenotypic and activation markers. Moreover, enzymatic digestion did not show higher overall yields of conventional dendritic cells and macrophages from the LN. Together, our findings show that the three dissociation protocols have similar impacts on the number and viability of cells isolated from the neonatal and adult LN. However, enzymatic digestion impacts the mean fluorescence intensity of key lineage and activation markers that may influence experimental findings.

Respiratory syncytial virus (RSV) invades the respiratory epithelium and provokes severe symptoms in young children leading to infant mortality. CD8 tissue resident memory (TRM) cells are associated with protection in adults against RSV, but are poorly established in neonatal mice consequent to primary infection. We hypothesized that antigen presentation contributes to age-dependent TRM establishment, thus we sought to define antigen kinetics in RSV-infected adult and neonatal mice. CB6F1/J adult and neonatal mice were infected with RSV and lung tissue were isolated over the course of acute and convalescent infection. Single cell lung suspension was co-cultured in vitrowith RSV K dM2 82–90(M2) or D bM 187–195(M) epitope-specific T cells isolated from CD8 T cell receptor (TCR) transgenic mice. T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) to quantify proliferative response 48 hours post-incubation at 37°C via flow cytometry. From lung tissue isolated at 7 days post infection (dpi), M and M2-specific CD8 T cells proliferated in response to co-culture; however, higher rates were induced by neonatal lung tissue primarily by M-specific T cells. By 8 and 9 dpi, proliferation induced by isolated neonatal lung continued while proliferation induced by adult lung tissue was not quantifiable by our assay. Our findings suggest antigen availability in neonates exceeds that of adults and is age and epitope-dependent. These discoveries indicate that antigen accessibility does not independentlycorrespond with the magnitude of the T cell response in early life. Exploring the inflammatory contributions to RSV-specific T cell defenses is required as this may aid in vaccine design and understanding disease pathogenesis. Supported by grant from NIH R01-AI154619.

Abstract Background Despite the high rate of respiratory syncytial virus (RSV) infection and severe symptoms in infants, subsequent infections occur, indicating impaired mucosal immune memory at early life. CD8 T cells are associated with reduced disease in human adult challenge trials and murine models, demonstrating their protective roles. However, since primary RSV infection occurs in infancy, a better understanding of the memory differentiation of RSV-specific respiratory CD8 T cells in neonates is required. Methods CB6F1/J mice were infected at 7, 14, 25 days of life or adulthood to characterize the RSV-specific memory T-cell development. Cells from mediastinal lymph nodes (MLN) and lungs were analyzed by flow cytometry at 7, 11, 14, and 40 days post RSV infection (dpi) after staining with fluorescent antibodies and tetramers specific for M and M2 epitopes in RSV. Results RSV-specific CD8 T cell responses were overall lower in neonates from peak to memory phase, but differ by epitope. At 40dpi, M-specific CD8 T cells preferentially differentiated to central-memory (CM) and resided in the MLN in both adult and neonatal mice, whereas M2-specific CD8 T cells preferentially differentiated to effector-memory (EM) and resided in the lungs in adult but not neonatal mice. Importantly, lung tissue resident memory cell differentiation was limited in early life and increased with age upon infection. Conclusion Our results indicate that the magnitude and memory development of RSV epitope-specific CD8 T cells differ by tissue and age at infection, suggesting that age-associated mucosal immune factors contribute to the development of RSV-specific memory T cells. This work was supported by funding through the NIAID R01AI154619 (A.M.W.M.). Supplemental support provided by USUHS Department of Pediatrics grant PED-86-3658 (A.M.W.M.).

Lipid nanoparticles (LNP) are essential components of messenger RNA (mRNA) vaccines that have been a cornerstone of the COVID-19 public health response. These LNPs are used for cell entry and protection of the mRNA. Others have shown that the LNP of the BNT162b2 vaccine is able to augment the immune response against the vaccine target. However, it is unknown how this LNP stimulates innate immune cells such as monocytes that participate in the engagement of the adaptive immune response. We used a THP-1 monocyte reporter cell line for nuclear factor kappa B (NF-κB) and interferon regulatory factor (IRF) activation to understand if empty LNPs could stimulate innate cells in comparison with toll-like receptor (TLR) agonists. With this cell line, we show that the BNT162b2’s LNP is able to initiate activation of NF-κB, but not IRF in a dose-dependent manner. The NF-κB response was similar to that of low doses of R848, a TLR 7/8 agonist, as well as LPS, a TLR4 agonist. Next, we evaluated NF-kB activation in the absence of MyD88 or TRIF using knockout reporter cell lines. We demonstrate that NF-κB activation is reduced in both knockout cell lines. In comparison, LPS primarily stimulated NF-kB activation through MyD88, and R848 relies on both pathways. Our data show that LNPs can stimulate key innate immune cells through NF-kB pathways but not through IRF suggesting that this master regulator may participate in the immune activation induced by the current mRNA vaccine platform. Moreover, our data suggest that LNPs can use both MyD88 and TRIF signaling cascades to induce activation. Understanding the mechanisms behind the immunostimulatory capacity of LNPs can shed light on modulating these vaccine components to enhance vaccine design. Defense Health Program, HU00012120094

This study demonstrates the impact of adjuvant on the development of T follicular helper (Tfh) and B cells, and their influence on antibody responses in mice vaccinated with SARS-CoV-2-spike-ferritin-nanoparticle (SpFN) adjuvanted with either Army Liposome Formulation containing QS-21 (SpFN + ALFQ) or Alhydrogel ® (SpFN + AH). SpFN + ALFQ increased the size and frequency of germinal center (GC) B cells in the vaccine-draining lymph nodes and increased the frequency of antigen-specific naive B cells. A single vaccination with SpFN + ALFQ resulted in a higher frequency of IL-21-producing-spike-specific Tfh and GC B cells in the draining lymph nodes and spleen, S-2P protein-specific IgM and IgG antibodies, and elicitation of robust cross-neutralizing antibodies against SARS-CoV-2 variants as early as day 7, which was enhanced by a second vaccination. This was associated with the generation of high titer, high avidity binding antibodies. The third vaccination with SpFN + ALFQ elicited high levels of neutralizing antibodies against the Omicron variant. No cross-neutralizing antibodies against Omicron were induced with SpFN + AH. These findings highlight the importance of ALFQ in orchestrating early induction of antigen-specific Tfh and GC B cell responses and long-lived plasma cells in the bone marrow. The early engagement of S-2P specific naive B cells and high titer IgM antibodies shape the development of long-term neutralization breadth.

Young infants frequently experience respiratory tract infections, yet vaccines designed to provide mucosal protection are lacking. Localizing pathogen-specific cellular and humoral immune responses to the lung could provide improved immune protection. We used a well-characterized murine model of respiratory syncytial virus (RSV) to study the development of lung-resident memory T cells (TRM) in neonatal compared to adult mice. We demonstrated that priming with RSV during the neonatal period failed to retain RSV-specific clusters of differentiation (CD8) TRM 6 weeks post infection, in contrast to priming during adulthood. The reduced development of RSV-specific TRM was associated with poor acquisition of two key markers of tissue residence: CD69 and CD103. However, by augmenting both innate immune activation and antigen exposure, neonatal RSV-specific CD8 T cells increased expression of tissue-residence markers and were maintained in the lung at memory time points. Establishment of TRM correlated with more rapid control of the virus in the lungs upon reinfection. This is the first strategy to effectively establish RSV-specific TRM in neonates providing new insight into neonatal memory T cell development and vaccine strategies.

Background Characterizing the longevity and quality of cellular immune responses to SARS-CoV-2 is critical to understanding immunologic approaches to protection against COVID-19. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Further analysis of the function, durability, and diversity of the cellular response long after natural infection, over a wider range of ages and disease phenotypes, is needed to further identify preventative and therapeutic interventions. Methods We identified participants in our multi-site longitudinal, prospective cohort study 12-months post SARS-CoV-2 infection representing a range of disease severity. We investigated the function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry. In parallel, the magnitude of SARS-CoV-2-specific antibodies was compared. Results SARS-CoV-2-specific antibodies and T cells were detected at 12-months post-infection. Severity of acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12-months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. Conclusions Our data show that SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12-months post-infection, with higher frequency noted in those who originally experienced severe disease.

Abstract Background The initial response of immune cells against respiratory viruses often determines the severity and duration of disease. The early trajectory of the immune response during infection with SARS-CoV-2 remains poorly understood. Dysregulation of innate immune factors that facilitate viral clearance and the adaptive response, such as type I interferons, have been implicated in severe COVID-19. However, collection of biological samples during the first seven days post-symptom onset has posed a logistical challenge, limiting our knowledge surrounding the immune responses that drive protection versus immunopathology. Methods From March 2020, Military Health System beneficiaries presenting with a positive SARS-CoV-2 test, a COVID-19 like illness, or a high-risk SARS-CoV-2 exposure at nine military medical treatment facilities across the United States were eligible for enrollment in our longitudinal cohort study, which included collection of respiratory sample, sera, plasma, and peripheral blood mononuclear cells (PBMCs). Twenty-five SARS-CoV-2 infected study participants provided samples with in the first seven days of symptom onset, fifteen of whom were hospitalized with COVID-19. We employed multiparameter spectral flow cytometry to comprehensively analyze the early trajectory of the innate and adaptive immune responses. Results We discovered that early activation of critical antigen presenting cell subsets was impaired upon comparing inpatients with outpatients, correlating with decreased antigen-experienced T cell responses. Specifically, we noted reduced expression of key costimulatory molecules, CD80 and CD86, on conventional dendritic cells that are required for viral antigen-specific T cell priming. Reduction in CD38, a marker of activation was also observed on inpatient dendritic cell subsets. Conclusion Reduced antigen presenting cell activation and expression of ligands that facilitate T cell engagement may impede the efficient clearance of SARS-CoV-2, coinciding with more severe disease in our cohort. Further analysis of the functional activation of early innate immune responses triggered by SARS-CoV-2 may unveil new immune biomarkers and therapeutic targets to predict and prevent severe disease associated with inadequate T cell immunity. Disclosures Simon Pollett, MBBS, Astra Zeneca (Other Financial or Material Support, HJF, in support of USU IDCRP, funded under a CRADA to augment the conduct of an unrelated Phase III COVID-19 vaccine trial sponsored by AstraZeneca as part of USG response (unrelated work))

Potent cellular responses to viral infections are pivotal for long -lived protection. Evidence is growing that these responses are critical in SARS -CoV-2 immunity. Assessment of a SARS -CoV-2 spike ferritin nanoparticle (SpFN) immunogen paired with two distinct adjuvants, Alhydrogel® (AH) or Army Liposome Formulation containing QS-21 (ALFQ) demonstrated unique vaccine evoked immune signatures. SpFN+ALFQ enhanced recruitment of highly activated classical and non -classical antigen presenting cells (APCs) to the vaccine-draining lymph nodes of mice. The multifaceted APC response of SpFN+ALFQ vaccinated mice was associated with an increased frequency of polyfunctional spike -specific T cells with a bias towards TH1 responses and more robust SARS-CoV-2 spike-specific recall response. In addition, SpFN+ALFQ induced Kb spike(539-546)-specific memory CD8+ T cells with effective cytolytic function and distribution to the lungs. This epitope is also present in SARS-CoV, thus suggesting that generation of cross-reactive T cells may provide protection against other coronavirus strains. Our study reveals that a nanoparticle vaccine, combined with a potent adjuvant, generates effective SARS-CoV-2 specific innate and adaptive immune T cell responses that are key components to inducing long-lived immunity. One Sentence Summary SpFN vaccine generates multifactorial cellular immune responses.

Young infants experience a higher burden of morbidity and mortality due to respiratory viruses and incomplete understanding of distinct age-dependent immune responses limits precision vaccine design in this age group. Subspecialized DCs and monocytes have evolved to detect viral pathogen-associated molecular patterns (PAMPs) to defend against infection and drive the adaptive immune response. We hypothesized that viral PAMPs and IFN I differentially activate neonatal DC and monocyte subsets resulting in age-dependent cytokine responses. To test this hypothesis, we isolated mononuclear cells from cord blood (CBMC) and peripheral blood (PBMC) and analyzed their responses through novel multiparameter spectral flow cytometric assays to PAMPs mimicking viral RNA. We discovered that conventional (c) DC2 in early life respond to Resiquimod (R848, TLR7/8 agonist) with similar upregulation of activation markers (CD80/86, CD70, CD38) and expression of TNFa, but more IL-6 compared to their adult counterparts. Unexpectedly, R848 and polyinosinic: polycytidylic acid (PIC, TLR3 agonist) resulted in significant CD25 upregulation by adult cDC2 and monocytes, in contrast to neonatal cells, suggesting decreased sensitivity to IL-2. In addition, in response to the antiviral type I IFN, cDC2 and monocytes from adults produced higher levels of CXCL10. Together our data suggest that neonatal DCs and monocytes have distinct functional responses to viral PAMPs, R848 and PIC, exhibiting reduced proinflammatory cytokine profiles. This may limit the potential for cytokine storm, but also delay the responsiveness to viral infections and vaccines that implement viral RNA in their design.

The emergence of variants of concern, some with reduced susceptibility to COVID-19 vaccines underscores consideration for the understanding of vaccine design that optimizes induction of effective cellular and humoral immune responses. We assessed a SARS-CoV-2 spike-ferritin nanoparticle (SpFN) immunogen paired with two distinct adjuvants, Alhydrogel ® or Army Liposome Formulation containing QS-21 (ALFQ) for unique vaccine evoked immune signatures. Recruitment of highly activated multifaceted antigen-presenting cells to the lymph nodes of SpFN+ALFQ vaccinated mice was associated with an increased frequency of polyfunctional spike-specific memory CD4 + T cells and K b spike-(539–546)-specific long-lived memory CD8 + T cells with effective cytolytic function and distribution to the lungs. The presence of this epitope in SARS-CoV, suggests that generation of cross-reactive T cells may be induced against other coronavirus strains. Our study reveals that a nanoparticle vaccine, combined with a potent adjuvant that effectively engages innate immune cells, enhances SARS-CoV-2-specific durable adaptive immune T cell responses.

We compare immunogenicity and protective efficacy of an HIV vaccine comprised of env and gag DNA and Env (Envelope) proteins by co-administration of the vaccine components in the same muscles or by separate administration of DNA + protein in contralateral sites in female rhesus macaques. The 6-valent vaccine includes gp145 Env DNAs, representing six sequentially isolated Envs from the HIV-infected individual CH505, and matching GLA-SE-adjuvanted gp120 Env proteins. Interestingly, only macaques in the co-administration vaccine group are protected against SHIV CH505 acquisition after repeated low-dose intravaginal challenge and show 67% risk reduction per exposure. Macaques in the co-administration group develop higher Env-specific humoral and cellular immune responses. Non-neutralizing Env antibodies, ADCC, and antibodies binding to FcγRIIIa are associated with decreased transmission risk. These data suggest that simultaneous recognition, processing, and presentation of DNA + Env protein in the same draining lymph nodes play a critical role in the development of protective immunity.

Abstract Background HIV sequence diversity and epitope immunodominance exerted by the variable regions are hurdles in the development of an effective AIDS vaccine. We developed novel DNA vaccine regimens to redirect T cell responses to highly conserved regions (CE) in the HIV proteome. Methods Rhesus macaques were primed with DNA expressing CE immunogen and boosted with a combination of DNAs expressing CE and/or full-length (FL) immunogens. Different prime-boost protocols were compared. The CE prime/CE+FL boost vaccination co-delivered in the same anatomical site was compared to delivery of CE and FL DNA in contralateral sites. Results CE prime/CE+FL boost regimens induced high and broad CTL responses which cannot be achieved using FL DNAs only. Thus, priming with CE DNA profoundly alters the immunodominance exerted by the variable non-CE regions, promoting responses to subdominant CE. This vaccine regimen is currently tested in clinical trials (HVTN 119, ACTG A5369). Comparison of same site vs contralateral vaccine delivery showed that separating CE and FL DNA resulted in increased breadth of T cell responses to non-CE while maintaining CTL responses to CE. Separating the immunogens at the prime allows for induction of independent primary adaptive responses by targeting the two vaccine components to distinct lymph nodes and preventing interference. Conclusions HIV DNA vaccine regimens comprising CE as prime and native immunogens as boost induce responses to subdominant epitopes, maximizing breadth and focusing the immune response against vulnerable regions of the virus. Separating the immunogens results in broader epitope recognition. This has implications for many vaccine protocols promoting induction of protective responses.

HIV sequence diversity and the propensity of eliciting immunodominant responses targeting non-essential variable regions are hurdles in the development of an effective AIDS vaccine. We developed a DNA vaccine comprising conserved elements (CE) of Gag and Env. Macaques (RM) received the CE DNA prime/CE+full-length DNA co-delivery booster vaccine regimen via intramuscular injection followed by electroporation. Priming vaccination with CE DNA is critical to efficiently overcome the dominance imposed by Gag and Env variable regions. CE vaccine induced broad, potent and durable cytotoxic T cell responses targeting conserved segments. Gag CE-specific T cells showed robust anamnestic responses upon infection with SIVmac239 which led to the identification of CE-specific cytotoxic lymphocytes able to recognize several CE epitopes presented on SIV infected cells in vivo. The T cell responses induced by the HIV Env CE immunogen were also recalled upon SIV infection, leading to the identification of two cross-reactive epitopes between HIV and SIV Env. These data demonstrate that the CE DNA vaccines changed the normal immunodominance patterns, eliciting immune responses that included subdominant, highly conserved epitopes. These vaccine regimens augment cytotoxic T cell responses to highly conserved epitopes in the viral proteome and maximize response breadth. The HIV Gag CE vaccine regimen is evaluated for prevention and immunotherapy in clinical trials (HVTN119 and ACTG 5369).

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide. It is unclear which arm of the immune system regulates resistance to HuNoV infection. Thus, we studied the pathogenesis of human norovirus (HuNoV) in T-B-NK+ Severe Combined Immunodeficiency (SCID) gnotobiotic pigs to investigate the role of innate (especially, natural killer (NK) cells) immunity in HuNoV infection. Forty SCID and non-SCID pigs were randomly grouped: 1) SCID + HuNoV (n = 12); 2) non-SCID + HuNoV (n = 14); 3) SCID mock-inoculated (n = 6); and 4) non-SCID mock-inoculated (n = 8). Pigs (8-14-day-old) were inoculated orally with GII.4 HuNoV strain HS292 (mean 9.1 log(10) genomic equivalents/pig) or mock. Daily fecal consistency and fecal viral RNA shedding, and histopathology (at euthanasia) were evaluated. Frequencies of blood and ileal T, B, and NK cells were analyzed by flow cytometry, and a NK cell cytotoxicity assay was performed at post-inoculation day (PID) 8. Unlike the increased infectivity of HuNoV observed previously in T-B-NK+ SCID pigs (Lei et al., 2016. Sci. Rep. 6, 25,222), there was no significant difference in frequency of pigs with diarrhea and diarrhea days between T-B-NK+ SCID + HuNoV and non-SCID + HuNoV groups. Cumulative fecal HuNoV RNA shedding at PIDs 1-8, PIDs 9-27, and PIDs 1-27 also did not differ statistically. These observations coincided with the presence of NK cells and NK cell cytotoxicity in the ileum and blood of the SCID pigs. Based on our observations, innate immunity, including NK cell activity, may be critical to mediate or reduce HuNoV infection in T-B-NK+ SCID pigs, and potentially in immunocompetent patients.