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Despite the success of global vaccination programs in slowing the spread of COVID-19, these efforts have been hindered by the emergence of new SARS-CoV-2 strains capable of evading prior immunity. The mutation and evolution of SARS-CoV-2 have created a demand for persistent efforts in vaccine development. SARS-CoV-2 Spike protein has been the primary target for COVID-19 vaccine development, but it is also the hotspot of mutations directly involved in host susceptibility and virus immune evasion. Our ability to predict emerging mutants and select conserved epitopes is critical for the development of a broadly neutralizing therapy or a universal vaccine. In this article, we review the general paradigm of immune responses to COVID-19 vaccines, highlighting the immunological epitopes of Spike protein that are likely associated with eliciting protective immunity resulting from vaccination in humans. Specifically, we analyze the structural and evolutionary characteristics of the SARS-CoV-2 Spike protein related to immune activation and function via the TLRs, B cells, and T cells. We aim to provide a comprehensive analysis of immune epitopes of Spike protein, thereby contributing to the development of new strategies for broad neutralization or universal vaccination., (Copyright © 2024 The Authors.)

Vaccines have demonstrated remarkable effectiveness in protecting against COVID-19; however, concerns regarding vaccine-associated enhanced respiratory diseases (VAERD) following breakthrough infections have emerged. Spike protein subunit vaccines for SARS-CoV-2 induce VAERD in hamsters, where aluminum adjuvants promote a Th2-biased immune response, leading to increased type 2 pulmonary inflammation in animals with breakthrough infections. To gain a deeper understanding of the potential risks and the underlying mechanisms of VAERD, we immunized ACE2-humanized mice with SARS-CoV-2 Spike protein adjuvanted with aluminum and CpG-ODN. Subsequently, we exposed them to increasing doses of SARS-CoV-2 to establish a breakthrough infection. The vaccine elicited robust neutralizing antibody responses, reduced viral titers, and enhanced host survival. However, following a breakthrough infection, vaccinated animals exhibited severe pulmonary immunopathology, characterized by a significant perivascular infiltration of eosinophils and CD4 + T cells, along with increased expression of Th2/Th17 cytokines. Intracellular flow cytometric analysis revealed a systemic Th17 inflammatory response, particularly pronounced in the lungs. Our data demonstrate that aluminum/CpG adjuvants induce strong antibody and Th1-associated immunity against COVID-19 but also prime a robust Th2/Th17 inflammatory response, which may contribute to the rapid onset of T cell-mediated pulmonary immunopathology following a breakthrough infection. These findings underscore the necessity for further research to unravel the complexities of VAERD in COVID-19 and to enhance vaccine formulations for broad protection and maximum safety., (© 2024 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Regulatory T cells that express the transcription factor Foxp3 (Treg cells) are a highly heterogenous population of immunoregulatory cells critical for maintaining immune homeostasis and preventing immunopathology during infections. Tissue resident Treg (TR-Treg) cells are maintained within nonlymphoid tissues and have been shown to suppress proinflammatory tissue resident T cell responses and promote tissue repair. Human populations are repetitively exposed to influenza infections and lung tissue resident effector T cell responses are associated with flu-induced long-term pulmonary sequelae. The kinetics of TR-Treg cell development and molecular features of TR-Treg cells during repeated and/or long-term flu infections are unclear. Utilizing a Foxp3 RFP /IL-10 GFP dual reporter mouse model along with intravascular fluorescent in vivo labeling, we characterized the TR-Treg cell responses to repetitive heterosubtypic influenza infections. We found lung tissue resident Treg cells accumulated and expressed high levels of co-inhibitory and co-stimulatory receptors post primary and secondary infections. Blockade of PD-1 or ICOS signaling reveals that PD-1 and ICOS signaling pathways counter-regulate TR-Treg cell expansion and IL-10 production, during secondary influenza infection. Furthermore, the virus-specific TR-Treg cell response displayed distinct kinetics, when compared to conventional CD4 + tissue resident memory T cells, during secondary flu infection. Our results provide insight into the tissue resident Foxp3 + regulatory T cell response during repetitive flu infections, which may be applicable to other respiratory infectious diseases such as tuberculosis and COVID., Competing Interests: WH received research support from MegaRobo Technologies Co., Ltd., which was not used in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McGee, Zhang, Magazine, Islam, Carossino and Huang.)

Type 1 regulatory T (Tr1) cells are an immunoregulatory CD4 + Foxp3- IL-10 high T cell subset with therapeutic potential for various inflammatory diseases. Retroviral (RV) transduction has been a valuable tool in defining the signaling pathways and transcription factors that regulate Tr1 differentiation and suppressive function. This protocol describes a method for RV transduction of naïve CD4 + T cells differentiating under Tr1 conditions, without the use of reagents such as polybrene or RetroNectin. A major advantage of this protocol over others is that it allows for the role of genes of interest on both differentiation and function of Tr1 cells to be interrogated. This is due to the high efficiency of RV transduction combined with the use of an IL10 GFP /Foxp3 RFP dual reporter mouse model, which enables successfully transduced Tr1 cells to be identified and sorted for functional assays. In addition, this protocol may be utilized for dual/multiple transduction approaches and transduction of other lymphocyte populations, such as CD8 + T cells., Competing Interests: Competing interests M.C.M. declares no conflict of interest. A patent disclosure was filed by W.H. and A.A. on the application of RAS signaling in Tr1 cell differentiation and function at Cornell University, when the original research on RAS signaling was published in 2017. W.H. received funding from MegaRobo Technologies, Ltd and A.A. received funding from 3M, which were not relevant to the research presented in this work., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Influenza (flu) infection is a leading cause of respiratory diseases and death worldwide. Although seasonal flu vaccines are effective at reducing morbidity and mortality, such effects rely on the odds of successful prediction of the upcoming viral strains. Additional threats from emerging flu viruses that we cannot predict and avian flu viruses that can be directly transmitted to humans urge the strategic development of universal vaccination that can protect against flu viruses of different subtypes and across species. Annual flu vaccines elicit mainly humoral responses. Under circumstances when antibodies induced by vaccination fail to recognize and neutralize the emerging virus adequately, virus-specific cytotoxic T lymphocytes (CTLs) are the major contributors to the control of viral replication and elimination of infected cells. Our studies exploited the evolutionary conservation of influenza A nucleoprotein (NP) and the fact that NP-specific CTL responses pose a constant selecting pressure on functional CTL epitopes to screen for NP epitopes that are highly conserved among heterosubtypes but are subjected to positive selection historically. We identified a region on NP that is evolutionarily conserved and historically positively selected (NP 137-182 ) and validated that it contains an epitope that is functional in eliciting NP-specific CTL responses and immunity that can partially protect immunized mice against lethal dose infection of a heterosubtypic influenza A virus. Our proof-of-concept study supports the hypothesis that evolutionary conservation and positive selection of influenza NP can be exploited to identify functional CTL epitope to elicit cross-protection against different heterosubtypes, therefore, to help develop strategies to modify flu vaccine formula for a broader and more durable protective immunity., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

The SARS-CoV-2 spike protein mediates target recognition, cellular entry, and ultimately the viral infection that leads to various levels of COVID-19 severities. Positive evolutionary selection of mutations within the spike protein has led to the genesis of new SARS-CoV-2 variants with greatly enhanced overall fitness. Given the trend of variants with increased fitness arising from spike protein alterations, it is critical that the scientific community understand the mechanisms by which these mutations alter viral functions. As of March 2022, five SARS-CoV-2 strains were labeled "variants of concern" by the World Health Organization: the Alpha, Beta, Gamma, Delta, and Omicron variants. This review summarizes the potential mechanisms by which the common mutations on the spike protein that occur within these strains enhance the overall fitness of their respective variants. In addressing these mutations within the context of the SARS-CoV-2 spike protein structure, spike/receptor binding interface, spike/antibody binding, and virus neutralization, we summarize the general paradigms that can be used to estimate the effects of future mutations along SARS-CoV-2 evolution.

Type 1 regulatory T (Tr1) cells can modulate inflammation through multiple direct and indirect molecular and cellular mechanisms and have demonstrated potential for anti-inflammatory therapies. Tr1 cells do not express the master transcription factor of conventional regulatory T cells, Foxp3, but express high levels of the immunomodulatory cytokine, IL-10. IL-2-inducible T-cell kinase (ITK) is conserved between mouse and human and is highly expressed in T cells. ITK signaling downstream of the T-cell receptor (TCR) is critical for T-cell subset differentiation and function. Upon activation by TCR, ITK is critical for Ras activation, leading to downstream activation of MAPKs and upregulation of IRF4, which further enable Tr1 cell differentiation and suppressive function. We summarize here the structure, signaling pathway, and function of ITK in T-cell lineage designation, with an emphasis on Tr1 cell development and function.

Bruton's tyrosine kinase (BTK) signaling is involved in innate immune responses and regulates the production of proinflammatory cytokines that can contribute to COVID-19 immunopathology. Clinical trials with BTK inhibitors in COVID-19 treatment have been proposed, and previous studies have attempted to investigate the therapeutic effects of ibrutinib and underlying mechanisms in treating viral pneumonia. These attempts, however, did not consider potential off target effect of BTK inhibitors on T cell differentiation, function, and survival, which may be beneficial in treatment for COVID-19. Here, we summarize the current knowledge of BTK/IL-2-inducible T-cell kinase (ITK) signaling in immunopathology and lymphopenia and discuss the potential of BTK/ITK dual inhibitors such as ibrutinib in modulating immunopathology and lymphopenia, for COVID-19 therapy., (©2020 Society for Leukocyte Biology.)

Adeno-associated viral (AAV) vectors have emerged as the leading gene delivery platform for gene therapy and vaccination. Three AAV-based gene therapy drugs, Glybera, LUXTURNA, and ZOLGENSMA were approved between 2012 and 2019 by the European Medicines Agency and the United States Food and Drug Administration as treatments for genetic diseases hereditary lipoprotein lipase deficiency (LPLD), inherited retinal disease (IRD), and spinal muscular atrophy (SMA), respectively. Despite these therapeutic successes, clinical trials have demonstrated that host anti-viral immune responses can prevent the long-term gene expression of AAV vector-encoded genes. Therefore, it is critical that we understand the complex relationship between AAV vectors and the host immune response. This knowledge could allow for the rational design of optimized gene transfer vectors capable of either subverting host immune responses in the context of gene therapy applications, or stimulating desirable immune responses that generate protective immunity in vaccine applications to AAV vector-encoded antigens. This review provides an overview of our current understanding of the AAV-induced immune response and discusses potential strategies by which these responses can be manipulated to improve AAV vector-mediated gene transfer., Competing Interests: Declaration of Competing Interest The authors declare no commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

CD4+ effector T cells effectuate T cell immune responses, producing cytokines to orchestrate the nature and type of immune responses. The non-receptor tyrosine kinase IL-2 inducible T cell kinase (ITK), a mediator of T cell Receptor signaling, plays a critical role in tuning the development of these effector cells. In this review we discussed the role that signals downstream of ITK, including the Ras/MAPK pathway, play in differentially controlling the differentiation of TH17, Foxp3+ T regulatory (Treg) cells, and Type 1 regulatory T (Tr1) cells, supporting a model of ITK signals controlling a decision point in the effector T cell differentiation process., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Interleukin-2 (IL-2) inducible T-cell kinase (ITK) is a non-receptor tyrosine kinase highly expressed in T-cell lineages and regulates multiple aspects of T-cell development and function, mainly through its function downstream of the T-cell receptor. Itk deficiency can lead to CD4 lymphopenia and Epstein-Bar virus (EBV)-associated lymphoproliferation and recurrent pulmonary infections in humans. However, the role of the ITK signaling pathway in pulmonary responses in active tuberculosis due to Mtb infection is not known. We show here that human lungs with active tuberculosis exhibit altered T-cell receptor/ITK signaling and that Itk deficiency impaired early protection against Mtb in mice, accompanied by defective development of IL-17A-producing γδ T cells in the lungs. These findings have important implications of human genetics associated with susceptibility to Mtb due to altered immune responses and molecular signals modulating host immunity that controls Mtb activity. Enhancing ITK signaling pathways may be an alternative strategy to target Mtb infection, especially in cases with highly virulent strains in which IL-17A plays an essential protective role., (Copyright © 2020 Huang, Ye, McGee, Nidetz, Elmore, Limper, Southard, Russell, August and Huang.)

Object: Stereotactic radiosurgery (SRS) alone is increasingly used in patients with newly diagnosed brain metastases. Stereotactic radiosurgery used together with whole-brain radiotherapy (WBRT) reduces intracranial failure rates, but this combination also causes greater neurocognitive toxicity and does not improve survival. Critics of SRS alone contend that deferring WBRT results in an increased need for salvage therapy and in higher costs. The authors compared the cost-effectiveness of treatment with SRS alone, SRS and WBRT (SRS+WBRT), and surgery followed by SRS (S+SRS) at the authors' institution., Methods: The authors retrospectively reviewed the medical records of 289 patients in whom brain metastases were newly diagnosed and who were treated between May 2001 and December 2007. Overall survival curves were plotted using the Kaplan-Meier method. Multivariate proportional hazards analysis (MVA) was used to identify factors associated with overall survival. Survival data were complete for 96.2% of patients, and comprehensive data on the resource use for imaging, hospitalizations, and salvage therapies were available from the medical records. Treatment costs included the cost of initial and all salvage therapies for brain metastases, hospitalizations, management of complications, and imaging. They were computed on the basis of the 2007 Medicare fee schedule from a payer perspective. Average treatment cost and average cost per month of median survival were compared. Sensitivity analysis was performed to examine the impact of variations in key cost variables., Results: No significant differences in overall survival were observed among patients treated with SRS alone, SRS+WBRT, or S+SRS with respective median survival of 9.8, 7.4, and 10.6 months. The MVA detected a significant association of overall survival with female sex, Karnofsky Performance Scale (KPS) score, primary tumor control, absence of extracranial metastases, and number of brain metastases. Salvage therapy was required in 43% of SRS-alone and 26% of SRS+WBRT patients (p < 0.009). Despite an increased need for salvage therapy, the average cost per month of median survival was $2412 per month for SRS alone, $3220 per month for SRS+WBRT, and $4360 per month for S+SRS (p < 0.03). Compared with SRS+WBRT, SRS alone had an average incremental cost savings of $110 per patient. Sensitivity analysis confirmed that the average treatment cost of SRS alone remained less than or was comparable to SRS+WBRT over a wide range of costs and treatment efficacies., Conclusions: Despite an increased need for salvage therapy, patients with newly diagnosed brain metastases treated with SRS alone have similar overall survival and receive more cost-effective care than those treated with SRS+WBRT. Compared with SRS+WBRT, initial management with SRS alone does not result in a higher average cost.

Purpose: Ionizing radiation, an important component of glioma therapy, is critically dependent on tumor oxygenation. However, gliomas are notable for areas of necrosis and hypoxia, which foster radioresistance. We hypothesized that pharmacologic manipulation of the typically dysfunctional tumor vasculature would improve intratumoral oxygenation and, thus, the antiglioma efficacy of ionizing radiation., Methods and Materials: Orthotopic U87 xenografts were treated with either continuous interferon-beta (IFN-beta) or bevacizumab, alone, or combined with cranial irradiation (RT). Tumor growth was assessed by quantitative bioluminescence imaging; the tumor vasculature using immunohistochemical staining, and tumor oxygenation using hypoxyprobe staining., Results: Both IFN-beta and bevaziumab profoundly affected the tumor vasculature, albeit with different cellular phenotypes. IFN-beta caused a doubling in the percentage of area of perivascular cell staining, and bevacizumab caused a rapid decrease in the percentage of area of endothelial cell staining. However, both agents increased intratumoral oxygenation, although with bevacizumab, the effect was transient, being lost by 5 days. Administration of IFN-beta or bevacizumab before RT was significantly more effective than any of the three modalities as monotherapy or when RT was administered concomitantly with IFN-beta or bevacizumab or 5 days after bevacizumab., Conclusion: Bevacizumab and continuous delivery of IFN-beta each induced significant changes in glioma vascular physiology, improving intratumoral oxygenation and enhancing the antitumor activity of ionizing radiation. Additional investigation into the use and timing of these and other agents that modify the vascular phenotype, combined with RT, is warranted to optimize cytotoxic activity.

Although temozolomide has shown clinical activity against neuroblastoma, this activity is likely limited by the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT). We hypothesized that IFN-beta could sensitize neuroblastoma cells to the cytotoxic effects of temozolomide through its ability to down-regulate MGMT expression. In vitro proliferation of three neuroblastoma cell lines treated with IFN-beta and temozolomide alone or in combination was examined. Antitumor activity was assessed in both localized and disseminated neuroblastoma xenografts using single-agent and combination therapy, with continuous delivery of IFN-beta being established by a liver-targeted adeno-associated virus-mediated approach. Two neuroblastoma cell lines (NB-1691 and SK-N-AS) were found to have high baseline levels of MGMT expression, whereas a third cell line (CHLA-255) had low levels. Temozolomide had little effect on in vitro proliferation of the neuroblastoma cell lines with high MGMT expression, but pretreatment with IFN-beta significantly decreased MGMT expression and cell counts (NB-1691: 36 +/- 3% of control, P = 0.0008; SK-N-AS: 54 +/- 7% control, P = 0.003). In vivo, NB-1691 tumors in CB17-SCID mice treated with the combination of IFN-beta and temozolomide had lower MGMT expression and a significantly reduced tumor burden, both localized [percent initial tumor volume: 2,516 +/- 680% (control) versus 1,272 +/- 330% (temozolomide), P = 0.01; 1,348 +/- 220%, P = 0.03 (IFN-beta); 352 +/- 110%, P = 0.0001 (combo)] and disseminated [bioluminescent signal: control (1.32e10 +/- 6.5e9) versus IFN-beta (2.78e8 +/- 3.09e8), P = 0.025, versus temozolomide (2.06e9 +/- 1.55e9), P = 0.1, versus combination (2.13e7 +/- 7.67e6), P = 0.009]. IFN-beta appears to sensitize neuroblastoma cells to the cytotoxic effects of temozolomide through attenuation of MGMT expression. Thus, IFN-beta and temozolomide may be a useful combination for treating children with this difficult disease.