Correction: Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

[This corrects the article DOI: 10.1371/journal.ppat.1009330.].

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

Simultaneous Aerosol and Intramuscular Immunization with Influenza Vaccine Induces Powerful Protective Local T Cell and Systemic Antibody Immune Responses in Pigs.

A vaccine providing both powerful Ab and cross-reactive T cell immune responses against influenza viruses would be beneficial for both humans and pigs. In this study, we evaluated i.m., aerosol (Aer), and simultaneous systemic and respiratory immunization (SIM) by both routes in Babraham pigs, using the single cycle candidate influenza vaccine S-FLU. After prime and boost immunization, pigs were challenged with H1N1pdm09 virus. i.m.-immunized pigs generated a high titer of neutralizing Abs but poor T cell responses, whereas Aer induced powerful respiratory tract T cell responses but a low titer of Abs. SIM pigs combined high Ab titers and strong local T cell responses. SIM showed the most complete suppression of virus shedding and the greatest improvement in pathology. We conclude that SIM regimes for immunization against respiratory pathogens warrant further study.

Establishment of a Pig Influenza Challenge Model for Evaluation of Monoclonal Antibody Delivery Platforms.

mAbs are a possible adjunct to vaccination and drugs in treatment of influenza virus infection. However, questions remain whether small animal models accurately predict efficacy in humans. We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing mAbs. We show that a strongly neutralizing mAb (2-12C) against the hemagglutinin head administered prophylactically at 15 mg/kg reduced viral load and lung pathology after pandemic H1N1 influenza challenge. A lower dose of 1 mg/kg of 2-12C or a DNA plasmid-encoded version of 2-12C reduced pathology and viral load in the lungs but not viral shedding in nasal swabs. We propose that the pig influenza model will be useful for testing candidate mAbs and emerging delivery platforms prior to human trials.

A randomized clinical study on the impact of Comprehensive Geriatric Assessment (CGA) based interventions on the quality of life of elderly, frail, onco-hematologic patients candidate to anticancer therapy: protocol of the ONCO-Aging study.

BACKGROUND: Age is considered as one of the most important risk-factor for many types of solid and hematological cancers, as their incidence increases with age in parallel to the ever-growing elderly population. Moreover, cancer incidence is constantly increasing as a consequence of the increase in life expectancy that favors the process of cellular senescence. Geriatric assessment has been increasingly recognized as predictive and prognostic instrument to detect frailty in older adults with cancer. In particular, the G8 score is a simple and reproducible instrument to identify elderly patients who should undergo full geriatric evaluation. Due to their frailty, elderly patients may be often under-treated and a therapeutic choice based also on a comprehensive geriatric assessment (CGA) is recommended. With these premises, we aim to test the impact of the CGA based interventions on the quality of life (QoL) of frail elderly onco-hematological patients, identified by the G8 screening, candidate for innovative target directed drugs or treatments including the combination of radiotherapy and chemotherapy (RT + CT). METHODS: Patients aged > 65 years, candidate to target directed agents or to RT + CT treatments are screened for frailty by the G8 test; those patients classified as frail (G8 ≤ 14) are randomized to receive a CGA at baseline or to conventional care. The primary endpoint is QoL, assessed by EORTC QLQ-C30C. As collateral biological study, the potential prognostic/predictive role of T-cell senescence and myeloid derived suppressor cells (MDSC) are evaluated on plasma samples. DISCUSSION: This trial will contribute to define the impact of CGA on the management of frail elderly onco-hematologic patients candidate to innovative biological drugs or to integrated schedules with the association of RT + CT. Furthermore, the use of plasma samples to assess the potential prognostic value of imbalance of immune-competent cells is expected to contribute to the individualized care of elderly patients, resulting into a fine tuning of the therapeutic strategies. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT04478916 . registered July 21, 2020 - retrospectively registered.

Inhibition of the Histone Methyltransferase EZH2 Enhances Protumor Monocyte Recruitment in Human Mesothelioma Spheroids.

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a long latency period and dismal prognosis. Recently, tazemetostat (EPZ-6438), an inhibitor of the histone methyltransferase EZH2, has entered clinical trials due to the antiproliferative effects reported on MPM cells. However, the direct and indirect effects of epigenetic reprogramming on the tumor microenvironment are hitherto unexplored. To investigate the impact of tumor-associated macrophages (TAMs) on MPM cell responsiveness to tazemetostat, we developed a three-dimensional MPM spheroid model that recapitulates in vitro, both monocytes' recruitment in tumors and their functional differentiation toward a TAM-like phenotype (Mo-TAMs). Along with an increased expression of genes for monocyte chemoattractants, inhibitory immune checkpoints, immunosuppressive and M2-like molecules, Mo-TAMs promote tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with tazemetostat enhances both the recruitment of Mo-TAMs and the expression of their protumor phenotype. Therefore, Mo-TAMs profoundly suppress the antiproliferative effects due to EZH2 inhibition in MPM cells. Overall, our findings indicate that TAMs are a driving force for MPM growth, progression, and resistance to tazemetostat; therefore, strategies of TAM depletion might be evaluated to improve the therapeutic efficacy of pharmacological inhibition of EZH2.

Insulin-like growth factor-1 receptor (IGF-1R) expression on circulating tumor cells (CTCs) and metastatic breast cancer outcome: results from the TransMYME trial.

PURPOSE: To evaluate the prognostic value of IGF-1R expression on circulating tumor cells (CTCs) in a prospective randomized clinical trial comparing chemotherapy plus metformin with chemotherapy alone in metastatic breast cancer (MBC) patients. METHODS: CTCs were collected at baseline and at the end of chemotherapy. An automated sample preparation and analysis system (CellSearch) were customized for detecting IGF-1R expression. The prognostic role of CTC count and IGF-1R was assessed for PFS and OS by univariate and multivariate analyses. RESULTS: Seventy-two out of 126 randomized patients were evaluated: 57% had ≥ 1 IGF-1R positive CTC and 37.5% ≥ 4 IGF-1R negative cells; 42% had CTC count ≥ 5/7.5 ml. At univariate analysis, the number of IGF-1R negative CTCs was strongly associated with risk of progression and death: HR 1.93 (P = 0.013) and 3.65 (P = 0.001), respectively; no association was detected between number of IGF-1R positive CTCs and PFS or OS (P = 0.322 and P = 0.840). The prognostic role of CTC count was confirmed: HR 1.69, P = 0.042 for PFS and HR 2.80 for OS, P = 0.002. By multivariate analysis, the prognostic role of the number of IGF-1R negative CTCs was maintained, while no residual prognostic role of CTC count or number of IGF-1R positive cells was found. CONCLUSION: Loss of IGF-1R in CTCs is associated with a significantly worse outcome in MBC patients. This finding supports further evaluation for the role of IGF-1R on CTCs to improve patient stratification and to implement new targeted strategies. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov (NCT01885013); European Clinical Trials Database (EudraCT No.2009-014,662-26).

HSP70/HSF1 axis, regulated via a PI3K/AKT pathway, is a druggable target in chronic lymphocytic leukemia.

Considering the role played by the heat shock protein of 70 kDa (HSP70) in cancer, we characterized this protein and its major regulator, the heat shock factor 1 (HSF1), in chronic lymphocytic leukemia (CLL). We found both HSP70 and HSF1 overexpressed in CLL patients, correlated to poor prognosis and abnormally localized in the nucleus of leukemic B cells. The two proteins were strictly correlated each other and their levels decreased consensually in those patients responding to in vivo therapeutic regimens. HSP70 and HSF1 inhibition was proved to be effective in inducing a dose-dependent in vitro apoptosis of CLL B cells. Considering that HSF1 is finely regulated by kinases belonging to pathways triggered by rat sarcoma (RAS), we benefited from a previous proteomic study performed in CLL patients aiming to assess the activation/expression of key signaling proteins. We found that patients showing high levels of HSP70 also expressed high Akt-Ser473, thus activating HSF1. Inhibition of PI3K, which activates AKT, reduced the expression of HSF1 and HSP70. By contrast, HSP70-low patients displayed high activation of MEK1/2 and ERK1/2, known to negatively regulate HSF1. These data demonstrate that the HSP70 expression is regulated by the modulation of HSF1 activity through the activation of RAS-regulated pathways and suggest the HSP70/HSF1 interplay as an interesting target for antileukemic therapies. Finally, inhibition of PI3K, that activates AKT, reduced the expression of HSF1 and HSP70.

Design of Gadoteridol-Loaded Cationic Liposomal Adjuvant CAF01 for MRI of Lung Deposition of Intrapulmonary Administered Particles.

Designing effective and safe tuberculosis (TB) subunit vaccines for inhalation requires identification of appropriate antigens and adjuvants and definition of the specific areas to target in the lungs. Magnetic resonance imaging (MRI) enables high spatial resolution, but real-time anatomical and functional MRI of lungs is challenging. Here, we describe the design of a novel gadoteridol-loaded cationic adjuvant formulation 01 (CAF01) for MRI-guided vaccine delivery of the clinically tested TB subunit vaccine candidate H56/CAF01. Gadoteridol-loaded CAF01 liposomes were engineered by using a quality-by-design approach to (i) increase the mechanistic understanding of formulation factors governing the loading of gadoteridol and (ii) maximize the loading of gadoteridol in CAF01, which was confirmed by cryotransmission electron microscopy. The encapsulation efficiency and loading of gadoteridol were highly dependent on the buffer pH due to strong attractive electrostatic interactions between gadoteridol and the cationic lipid component. Optimal gadoteridol loading of CAF01 liposomes showed good in vivo stability and safety upon intrapulmonary administration into mice while generating 1.5-fold MRI signal enhancement associated with approximately 30% T1 relaxation change. This formulation principle and imaging approach can potentially be used for other mucosal nanoparticle-based formulations, species, and lung pathologies, which can readily be translated for clinical use.

Cortactin, a Lyn substrate, is a checkpoint molecule at the intersection of BCR and CXCR4 signalling pathway in chronic lymphocytic leukaemia cells.

Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP-9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP-9 release as well as a decrease of migration capability of leukaemic B cells in vitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase-inhibitor PP2 with a consequent decrease of MMP-9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both in vivo and in vitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check-point molecule between the BCR and CXCR4 signalling pathways.

Lyn-mediated procaspase 8 dimerization blocks apoptotic signaling in B-cell chronic lymphocytic leukemia.

Lyn, a member of the group of tyrosine kinases named the Src family kinases (SFKs), is overexpressed, associated with an aberrant multiprotein complex and constitutively active in B-cell chronic lymphocytic leukemia (B-CLL) cells, resulting in a high level of tyrosine phosphorylation and contributing to their resistance to apoptosis. By using biochemical and bioinformatics tools, we identified procaspase-8 (procasp8), the caspase-8 zymogen, as a cytosolic target for Lyn in B-CLL cells, the phosphorylation of which at Tyr380 promotes the formation of an inactive procasp8 homodimer. This complex remains segregated in the cytosol and appears to be crucial in mediating the antiapoptotic function of Lyn in this disease. The significance of the Lyn-procasp8 axis in impairing apoptosis in B-CLL cells was further confirmed by pharmacological and genetic inhibition of procasp8, which drastically reduced the apoptosis induced by the SFK inhibitors PP2 and dasatinib. Our data highlight that Lyn's dysregulated expression, activity, and localization in B-CLLs support resistance to cell demise by inhibiting an early player of apoptotic signaling, and potentially broaden the perspectives of developing new strategies for the treatment of this disease.

Intrinsic and extrinsic mechanisms contribute to maintain the JAK/STAT pathway aberrantly activated in T-type large granular lymphocyte leukemia.

The JAK/STAT pathway is altered in T-cell large granular lymphocytic leukemia. In all patients, leukemic LGLs display upregulation of phosphorylated STAT3 (P-STAT3) that activates expression of many antiapoptotic genes. To investigate the mechanisms maintaining STAT3 aberrantly phosphorylated using transcriptional protein and functional assays, we analyzed interleukin (IL)-6 and suppressor of cytokine signaling-3 (SOCS3), 2 key factors of the JAK/STAT pathway that induce and inhibit STAT3 activation, respectively. We showed that IL-6 was highly expressed and released by the patients' peripheral blood LGL-depleted population, accounting for a trans-signaling process. By neutralizing IL-6 or its specific receptor with specific antibodies, a significant reduction of P-STAT3 levels and, consequently, LGL survival was demonstrated. In addition, we found that SOCS3 was down-modulated in LGL and unresponsive to IL-6 stimulation. By treating neoplastic LGLs with a demethylating agent, IL-6-mediated SOCS3 expression was restored with consequent P-STAT3 and myeloid cell leukemia-1 down-modulation. Methylation in the SOCS3 promoter was not detectable, suggesting that an epigenetic inhibition mechanism occurs at a different site. Our data indicate that loss of the inhibitor SOCS3 cooperates with IL-6 to maintain JAK/STAT pathway activation, thus contributing to leukemic LGL survival, and suggest a role of demethylating agents in the treatment of this disorder.

Ex Vivo Signaling Protein Mapping in T Lymphocytes in the Psoriatic Arthritis Joints.

We assessed signaling protein mapping in total T cells, to analyze the proportions of T regulatory (Treg) and TCD4+ effector (Teff) cell phenotypes, and the respective interleukin 6Rα (IL-6Rα) expression in the inflammatory microenvironment of synovial fluid (SF) of patients with sustained psoriatic arthritis (PsA). Our approach was to measure the IL-6 level in SF using a multiplex bead immunoassay. Reverse-phase protein array was used to assess Janus kinase (JAK) 1 and JAK2, extra-cellular regulated kinase (ERK) 1 and 2, protein kinase Cδ (PKCδ), signal transducer and activator and transcription (STAT) 1, STAT3, and STAT5 phosphoproteins in total T cell lysates from SF of patients with PsA. Frequencies of CD4+IL-17A-F+IL-23+ CD4+ Th cells producing IL-17A and IL-17F (Th17) and CD4+CD25high intracellular forkhead box transcription factor+ (FOXP3+) phenotypes, and the percentage of Treg- and Teff- cells were quantified in SF and matched peripheral blood (PB) of patients with PsA and PB of healthy controls (HC) by flow cytometry. Our results were the following: In PsA SF samples, a coordinate increase of JAK1, ERK1/2, STAT1, STAT3, and STAT5 phosphoproteins was found in total T cells in SF of PsA; where IL-6 levels were higher than in PB from HC. Expanded CD4+IL-17A-F+IL-23+ Th17, CD4+ CD25- Teff- and CD4+CD25(high) FoxP3+Treg subsets, showing similar levels of enhanced IL-6Rδ expression, were confined to PsA joints. In our studies, the transcriptional network profile identified by ex vivo signaling protein mapping in T lymphocytes in PsA joints revealed the complex interplay between IL-1, IL-6, and IL-23 signaling and differentiation of Th17 cells and CD4+Tregs in sustained joint inflammation in PsA.

Leukaemic cells from chronic lymphocytic leukaemia patients undergo apoptosis following microtubule depolymerization and Lyn inhibition by nocodazole.

Functional abnormalities of chronic lymphocytic leukaemia (CLL) cells may be related to the microtubular network of cell cytoskeleton; specifically tubulin involvement in cells after B-cell receptor engagement. As microtubule inhibitors could represent a therapeutic strategy for CLL, this study investigated the capability of nocodazole, a synthetic depolymerizing agent, to kill CLL leukaemic cells. We demonstrated that nocodazole was highly specific for the in vitro induction of apoptosis in leukaemic cells from 90 CLL patients, without affecting the viability of T-cells and/or mesenchymal stromal cells (MSCs) recovered from the same patients. Nocodazole was observed to overcome the pro-survival signals provided by MSCs. Competing with ATP for the nucleotide-binding site, nocodazole has been observed to turn off the high basal tyrosine phosphorylation of leukaemic cells mediated by the Src-kinase Lyn. Considering that most anti-microtubule drugs have limited clinical use because of their strong toxic effects, the high selectivity of nocodazole for leukaemic cells in CLL and its capability to bypass microenvironmental pro-survival stimuli, suggests the use of this inhibitor for designing new therapeutic strategies in CLL treatment.

Cortactin, another player in the Lyn signaling pathway, is over-expressed and alternatively spliced in leukemic cells from patients with B-cell chronic lymphocytic leukemia.

Cortactin, an actin binding protein and Lyn substrate, is up-regulated in several cancers and its level is associated with increased cell migration, metastasis and poor prognosis. The identification that the Src kinase Lyn and its substrate HS1 are over-expressed in B-cell chronic lymphocytic leukemia and involved in resistance to chemotherapy and poor prognosis, prompted us to investigate the role of cortactin, an HS1 homolog, in the pathogenesis and progression of this disorder. In this study, we observed that cortactin is over-expressed in leukemic cells of patients (1.10 ± 0.12) with respect to normal B lymphocytes (0.19 ± 0.06; P=0.0065). Fifty-three percent of our patients expressed the WT mRNA and p80/85 protein isoforms, usually lacking in normal B lymphocytes which express the SV1 variant and the p70/75 protein isoforms. Moreover, we found an association of the cortactin overexpression and negative prognostic factors, including ZAP-70 (P<0.01), CD38 (P<0.01) and somatic hypermutations in the immunoglobulin heavy-chain variable region (P<0.01). Our results show that patients with B-cell chronic lymphocytic leukemia express high levels of cortactin with a particular overexpression of the WT isoform that is lacking in normal B cells, and a correlation to poor prognosis, suggesting that this protein could be relevant in the pathogenesis and aggressiveness of the disease.

Detection of monoclonal T populations in patients with KIR-restricted chronic lymphoproliferative disorder of NK cells.

The etiology of chronic large granular lymphocyte proliferations is largely unknown. Although these disorders are characterized by the expansion of different cell types (T and natural killer) with specific genetic features and abnormalities, several lines of evidence suggest a common pathogenetic mechanism. According to this interpretation, we speculated that in patients with natural killer-type chronic lymphoproliferative disorder, together with natural killer cells, also T lymphocytes undergo a persistent antigenic pressure, possibly resulting in an ultimate clonal T-cell selection. To strengthen this hypothesis, we evaluated whether clonal T-cell populations were detectable in 48 patients with killer immunoglobulin-like receptor-restricted natural killer-type chronic lymphoproliferative disorder. At diagnosis, in half of the patients studied, we found a clearly defined clonal T-cell population, despite the fact that all cases presented with a well-characterized natural killer disorder. Follow-up analysis confirmed that the TCR gamma rearrangements were stable over the time period evaluated; furthermore, in 7 patients we demonstrated the appearance of a clonal T subset that progressively matures, leading to a switch between killer immunoglobulin-like receptor-restricted natural killer-type disorder to a monoclonal T-cell large granular lymphocytic leukemia. Our results support the hypothesis that a common mechanism is involved in the pathogenesis of these disorders.

Patients with ankylosing spondylitis present a distinct CD8 T cell subset with osteogenic and cytotoxic potential.

OBJECTIVES: Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease affecting mainly the axial skeleton. Peripheral involvement (arthritis, enthesitis and dactylitis) and extra-musculoskeletal manifestations, including uveitis, psoriasis and bowel inflammation, occur in a relevant proportion of patients. AS is responsible for chronic and severe back pain caused by local inflammation that can lead to osteoproliferation and ultimately spinal fusion. The association of AS with the human leucocyte antigen-B27 gene, together with elevated levels of chemokines, CCL17 and CCL22, in the sera of patients with AS, led us to study the role of CCR4+ T cells in the disease pathogenesis. METHODS: CD8+CCR4+ T cells isolated from the blood of patients with AS (n=76) or healthy donors were analysed by multiparameter flow cytometry, and gene expression was evaluated by RNA sequencing. Patients with AS were stratified according to the therapeutic regimen and current disease score. RESULTS: CD8+CCR4+ T cells display a distinct effector phenotype and upregulate the inflammatory chemokine receptors CCR1, CCR5, CX3CR1 and L-selectin CD62L, indicating an altered migration ability. CD8+CCR4+ T cells expressing CX3CR1 present an enhanced cytotoxic profile, expressing both perforin and granzyme B. RNA-sequencing pathway analysis revealed that CD8+CCR4+ T cells from patients with active disease significantly upregulate genes promoting osteogenesis, a core process in AS pathogenesis. CONCLUSIONS: Our results shed light on a new molecular mechanism by which T cells may selectively migrate to inflammatory loci, promote new bone formation and contribute to the pathological ossification process observed in AS.

The chemokine landscape: one system multiple shades.

Leukocyte trafficking is mainly governed by chemokines, chemotactic cytokines, which can be concomitantly produced in tissues during homeostatic conditions or inflammation. After the discovery and characterization of the individual chemokines, we and others have shown that they present additional properties. The first discoveries demonstrated that some chemokines act as natural antagonists on chemokine receptors, and prevent infiltration of leukocyte subsets in tissues. Later on it was shown that they can exert a repulsive effect on selective cell types, or synergize with other chemokines and inflammatory mediators to enhance chemokine receptors activities. The relevance of the fine-tuning modulation has been demonstrated in vivo in a multitude of processes, spanning from chronic inflammation to tissue regeneration, while its role in the tumor microenvironment needs further investigation. Moreover, naturally occurring autoantibodies targeting chemokines were found in tumors and autoimmune diseases. More recently in SARS-CoV-2 infection, the presence of several autoantibodies neutralizing chemokine activities distinguished disease severity, and they were shown to be beneficial, protecting from long-term sequelae. Here, we review the additional properties of chemokines that influence cell recruitment and activities. We believe these features need to be taken into account when designing novel therapeutic strategies targeting immunological disorders.

Effect of mucosal adjuvant IL-1ß on heterotypic immunity in a pig influenza model.

T cell responses directed against highly conserved viral proteins contribute to the clearance of the influenza virus and confer broadly cross-reactive and protective immune responses against a range of influenza viruses in mice and ferrets. We examined the protective efficacy of mucosal delivery of adenoviral vectors expressing hemagglutinin (HA) and nucleoprotein (NP) from the H1N1 virus against heterologous H3N2 challenge in pigs. We also evaluated the effect of mucosal co-delivery of IL-1ß, which significantly increased antibody and T cell responses in inbred Babraham pigs. Another group of outbred pigs was first exposed to pH1N1 as an alternative means of inducing heterosubtypic immunity and were subsequently challenged with H3N2. Although both prior infection and adenoviral vector immunization induced strong T-cell responses against the conserved NP protein, none of the treatment groups demonstrated increased protection against the heterologous H3N2 challenge. Ad-HA/NP+Ad-IL-1ß immunization increased lung pathology, although viral load was unchanged. These data indicate that heterotypic immunity may be difficult to achieve in pigs and the immunological mechanisms may differ from those in small animal models. Caution should be applied in extrapolating from a single model to humans.