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Despite all successful efforts to develop a COVID-19 vaccine, the need to evaluate alternative antigens to produce next-generation vaccines is imperative to target emerging variants. Thus, the second generation of COVID-19 vaccines employ more than one antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce an effective and lasting immune response. Here, we analyzed the combination of two SARS-CoV-2 viral antigens that could elicit a more durable immune response in both T- and B-cells. The nucleocapsid (N) protein, Spike protein S1 domain, and receptor binding domain (RBD) of the SARS-CoV-2 spike surface glycoproteins were expressed and purified in a mammalian expression system, taking into consideration the posttranscriptional modifications and structural characteristics. The immunogenicity of these combined proteins was evaluated in a murine model. Immunization combining S1 or RBD with the N protein induced higher levels of IgG antibodies, increased the percentage of neutralization, and elevated the production of cytokines TNF-α, IFN-γ, and IL-2 compared to the administration of a single antigen. Furthermore, sera from immunized mice recognized alpha and beta variants of SARS-CoV-2, which supports ongoing clinical results on partial protection in vaccinated populations, despite mutations. This study identifies potential antigens for second-generation COVID-19 vaccines.

Over a century ago, bacterial extracts were found to be useful in cancer therapy, but this treatment modality was obviated for decades. Currently, in spite of the development and advances in chemotherapies and radiotherapy, failure of these conventional treatments still represents a major issue in the complete eradication of tumor cells and has led to renewed approaches with bacteria-based tumor therapy as an alternative treatment. In this context, live-attenuated bacteria, particularly Salmonella enterica , have demonstrated tumor selectivity, intrinsic oncolytic activity, and the ability to induce innate or specific antitumor immune responses. Moreover, Salmonella enterica also has strong potential as a delivery system of tumor-associated antigens, cytotoxic molecules, immunomodulatory molecules, pro-apoptotic proteins, and nucleic acids into eukaryotic cells, in a process known as bactofection and antitumor nanoparticles. In this review, we present the state of the art of current preclinical and clinical research on the use of Salmonella enterica as a potential therapeutic ally in the war against cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.

Cancer remains a public health problem worldwide. Although conventional therapies have led to some excellent outcomes, some patients fail to respond to treatment, they have few therapeutic alternatives and a poor survival prognosis. Several strategies have been proposed to overcome this issue. The most recent approach is immunotherapy, particularly the use of recombinant antibodies and their derivatives, such as the single-chain fragment variable (scFv) containing the complete antigen-binding domains of a whole antibody that successfully targets tumor cells. This review describes the recent progress made with scFvs as a cancer diagnostic and therapeutic tool, with an emphasis on preclinical approaches and their potential use in clinical trials.

Purpose: Rheumatoid arthritis (RA) and primary Sjögren's syndrome (pSS) are autoimmune diseases (ADs) characterized by joint damage and involvement of the salivary glands, respectively. ADs share some susceptibility loci, such as TNFSF4, which is a classical susceptibility gene associated with systemic lupus erythematosus, but its role in RA and pSS is not yet clear. Thus, the aim of this study was to determine whether three TNFSFS4 polymorphisms are associated with RA and pSS., Methods: Our case-control study included 500 controls, 459 patients with RA, and 210 patients with pSS from Mexico. TNFSF4 single nucleotide polymorphisms (SNPs) rs1234315C/T, rs2205960G/T, and rs704840T/G were genotyped using TaqMan probes and discrimination allelic assay., Results: The three TNFSF4 SNPs were associated with susceptibility to RA (rs1234315C/T: odds ratio [OR] 1.4, p = 0.01; rs2205960G/T: OR 1.23, p = 0.03; rs704840T/G: OR 1.24, p = 0.02). An association between TNFSF4 rs1234315C/T and pSS was also observed (OR 1.28, p = 0.04), however, after Bonferroni correction, this association was lost., Conclusion: Our data suggest that TNFSF4 could be a risk factor in RA but not pSS in a Mexican population., (Published by Elsevier GmbH.)

The Flaviviridae are a family of positive-sense, single-stranded RNA enveloped viruses, and their members belong to a single genus, Flavivirus. Flaviviruses are found in mosquitoes and ticks; they are etiological agents of: dengue fever, Japanese encephalitis, West Nile virus infection, Zika virus infection, tick-borne encephalitis, and yellow fever, among others. Only a few flavivirus vaccines have been licensed for use in humans: yellow fever, dengue fever, Japanese encephalitis, tick-borne encephalitis, and Kyasanur forest disease. However, improvement is necessary in vaccination strategies and in understanding of the immunological mechanisms involved either in the infection or after vaccination. This is especially important in dengue, due to the immunological complexity of its four serotypes, cross-reactive responses, antibody-dependent enhancement, and immunological interference. In this context, mucosal vaccines represent a promising alternative against flaviviruses. Mucosal vaccination has several advantages, as inducing long-term protective immunity in both mucosal and parenteral tissues. It constitutes a friendly route of antigen administration because it is needle-free and allows for a variety of antigen delivery systems. This has promoted the development of several ways to stimulate immunity through the direct administration of antigens (e.g., inactivated virus, attenuated virus, subunits, and DNA), non-replicating vectors (e.g., nanoparticles, liposomes, bacterial ghosts, and defective-replication viral vectors), and replicating vectors (e.g., Salmonella enterica , Lactococcus lactis , Saccharomyces cerevisiae , and viral vectors). Because of these characteristics, mucosal vaccination has been explored for immunoprophylaxis against pathogens that enter the host through mucosae or parenteral areas. It is suitable against flaviviruses because this type of immunization can stimulate the parenteral responses required after bites from flavivirus-infected insects. This review focuses on the advantages of mucosal vaccine candidates against the most relevant flaviviruses in either humans or animals, providing supporting data on the feasibility of this administration route for future clinical trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2022 Luria-Pérez, Sánchez-Vargas, Muñoz-López and Mellado-Sánchez.)

Arnica montana cell suspension culture could be a sustainable source of a vegetal material producer of secondary metabolites (SMs) possessing biological effects. Different plant growth regulator concentrations (0-5 mg/L) were tested in foliar explants to induce a callus that was used to establish a cell suspension culture. Growth kinetics was carried out for 30 days. A methanolic extract obtained from biomass harvested at 30 days of growth kinetics was fractionated, and three fractions were tested for bioactivities. We induced a callus with 1 mg/L of picloram and 0.5 mg/L of kinetin in foliar explants, which allowed for the establishment of a cell suspension culture, and the latter had the highest total SMs contents at day 30. Three fractions showed differences in total SMs contents, with the highest values per gram as follows: 270 mg gallic acid equivalent for total phenolic content, 200 mg quercetin equivalent for total flavonoid content, 83 mg verbascoside equivalent for total phenolic acid content, and 396 mg parthenolide equivalent for total sesquiterpene lactone content. The best bioactivities were 2-6 µg/mL for the 50% inhibition of 2,2-diphenyl-1-picrylhydrazyl radical, 30% cellular viability of lymphoma cells at 40 µg/mL, 17% inhibition against Escherichia coli and Staphylococcus aureus at 8 µg/disk, and α -amylase inhibition at 12% with 10 µg/mL. The total SMs contents were correlated with bioactivities.

Hematologic malignancies such as leukemias and lymphomas are among the leading causes of pediatric cancer death worldwide, and although survival rates have improved with conventional treatments, the development of drug-resistant cancer cells may lead to patient relapse and limited possibilities of a cure. Drug-resistant cancer cells in these hematologic neoplasms are induced by overexpression of the antiapoptotic B-cell lymphoma 2 (Bcl-2) protein families, such as Bcl- XL , Bcl-2, and Mcl-1. We have previously shown that peptides from the BH3 domain of the proapoptotic Bax protein that also belongs to the Bcl-2 family may antagonize the antiapoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. Furthermore, cell-permeable Bax BH3 peptides also elicit antitumor activity and extend survival in a murine xenograft model of human B non-Hodgkin's lymphoma. However, the activity of the BH3 peptides of the proapoptotic Bak protein of the Bcl-2 family against these hematologic malignant cells requires further characterization. In this study, we report the ability of the cell-permeable Bak BH3 peptide to restore apoptosis and induce chemosensitization of acute lymphoblastic leukemia and non-Hodgkin's lymphoma cell lines, and this event is enhanced with the coadministration of cell-permeable Bax BH3 peptide and represents an attractive approach to improve the patient outcomes with relapsed or refractory hematological malignant cells., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Omar Ugarte-Alvarez et al.)

The survival of patients with non-Hodgkin's lymphoma (NHL) has substantially improved with current treatments. Nevertheless, the appearance of drug-resistant cancer cells leads to patient relapse. It is therefore necessary to find new antitumor therapies that can completely eradicate transformed cells. Chemotherapy-resistant cancer cells are characterized by the overexpression of members of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein family, such as Bcl- XL , Bcl-2, and Mcl-1. We have recently shown that peptides derived from the BH3 domain of the pro-apoptotic Bax protein may antagonize the anti-apoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. In this study, we investigated the feasibility of releasing this peptide into the tumor microenvironment using live attenuated Salmonella enterica , which has proven to be an ally in cancer therapy due to its high affinity for tumor tissue, its ability to activate the innate and adaptive antitumor immune responses, and its potential use as a delivery system of heterologous molecules. Thus, we expressed and released the cell-permeable Bax BH3 peptide from the surface of Salmonella enterica serovar Typhimurium SL3261 through the MisL autotransporter system. We demonstrated that this recombinant bacterium significantly decreased the viability and increased the apoptosis of Ramos cells, a human B NHL cell line. Indeed, the intravenous administration of this recombinant Salmonella enterica elicited antitumor activity and extended survival in a xenograft NHL murine model. This antitumor activity was mediated by apoptosis and an inflammatory response. Our approach may represent an eventual alternative to treat relapsing or refractory NHL., (Copyright © 2019 Mateos-Chávez, Muñoz-López, Becerra-Báez, Flores-Martínez, Prada-Gracia, Moreno-Vargas, Baay-Guzmán, Juárez-Hernández, Chávez-Munguía, Cabrera-Muñóz and Luria-Pérez.)

Worldwide, neoplasms of the gastrointestinal tract have a very high incidence and mortality. Among these, colorectal cancer, which includes colon and rectum malignancies, representing both highest incidence and mortality. While gallbladder cancer, another neoplasm associated to gastrointestinal tract occurs less frequently. Genetic factors, inflammation and nutrition are important risk factors associated with colorectal cancer development. Likewise, pathogenic microorganisms inducing intestinal dysbiosis have become an important scope to determine the role of bacterial infection on tumorigenesis. Interestingly, in human biopsies of different types of gastrointestinal tract cancer, the presence of different bacterial strains, such as Fusobacterium nucleatum , Escherichia coli , Bacteroides fragilis and Salmonella enterica have been detected, and it has been considered as a high-risk factor to cancer development. Therefore, pathogens infection could contribute to neoplastic development through different mechanisms; including intestinal dysbiosis, inflammation, evasion of tumoral immune response and activation of pro-tumoral signaling pathways, such as β catenin. Here, we have reviewed the suggested bacterial molecular mechanisms and their possible role on development and progression of gastrointestinal neoplasms, focusing mainly on colon neoplasms, where the bacteria Fusobacterium nucleatum , Escherichia coli , Bacteroides fragilis and Salmonella enterica infect., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2019 Marco Antonio Hernández-Luna et al.)

An antibody-cytokine fusion protein, composed of the murine single-chain cytokine interleukin-12 (IL-12) genetically fused to a human IgG3 specific for the human tumor-associated antigen HER2/neu maintains antigen binding, cytokine bioactivity, and IL-12 heparin-binding activity. This latter property is responsible for the binding of the cytokine to glycosaminoglycans (GAGs) on the cell surface and the extracellular matrix and has been implicated in modulating IL-12 bioactivity. Previous studies indicate that the p40 subunit of human and murine IL-12 is responsible for the heparin-binding activity of this heterodimeric cytokine. In the present study we used bioinformatic analysis and site-directed mutagenesis to develop a version of the antibody-(IL-12) fusion protein without heparin-binding activity. This was accomplished by replacing the basic arginine (R) and lysine (K) residues in the cluster of amino acids 254-260 (RKKEKMK) of the murine IL-12 p40 subunit by the neutral non-polar amino acid alanine (A), generating an AAAEAMA mutant fusion protein. ELISA and flow cytometry demonstrated that the antibody fusion protein lacks heparin-binding activity but retains antigen binding. A T-cell proliferation assay showed IL-12 bioactivity in this construct. However, the IL-12 bioactivity is decreased compared to its non-mutated counterpart, which is consistent with an ancillary role of the heparin-binding site of IL-12 in modulating its activity. Thus, we have defined a cluster of amino acid residues with a crucial role in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein., (Copyright © 2019. Published by Elsevier Ltd.)

In recent years, cancer immunotherapy has undergone great advances because of our understanding of the immune response and the mechanisms through which tumor cells evade it. A century after the first immunotherapy attempt based on bacterial products described by William Coley, the use of live attenuated bacterial vectors has become a promising alternative in the fight against cancer. This review describes the role of live attenuated Salmonella enterica as an oncolytic and immunotherapeutic agent, due to its high affinity for tumor tissue and its ability to activate innate and adaptive antitumor immune response. Furthermore, its potential use as delivery system of tumor antigens and immunomodulatory molecules that induce tumor regression is also reviewed.

Background: Tumor cell resistance to chemotherapy agents is one of the main problems in the eradication of different neoplasias. One of the mechanisms of this process is the overexpression of anti-apoptotic proteins such as Bcl-2 and Bcl- XL ; blocking the activity of these proteins may contribute to the sensitization of tumor cells and allow the adequate effects of chemotherapeutic drugs., Methods and Results: This study adressed the transfection of prostate cancer cells (PC3) with a plasmid encoding a recombinant protein with an antagonist peptide from the BH3 region of the Bax protein fused to the GFP reporter protein (BaxGFP). This protein induced apoptosis of these tumor cells; further, selective transport of this plasmid to the tumor cell with Salmonella enterica serovar Typhimurium (strain SL3261), a live-attenuated bacterial vector, can induce sensitization of the tumor cell to the action of drugs such as cisplatin, through a process known as bactofection., Conclusions: These results suggest that Salmonella enterica can be used as a carrier vector of nucleotide sequences encoding heterologous molecules used in antitumor therapy., (Copyright © 2016 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.)

Iron is essential for cell growth and is imported into cells in part through the action of transferrin (Tf), a protein that binds its receptor (TfR1 or CD71) on the surface of a cell, and then releases iron into endosomes. TfR1 is a single pass type-II transmembrane protein expressed at basal levels in most tissues. High expression of TfR1 is typically associated with rapidly proliferating cells, including various types of cancer. TfR1 is targeted by experimental therapeutics for several reasons: its cell surface accessibility, constitutive endocytosis into cells, essential role in cell growth and proliferation, and its overexpression by cancer cells. Among the therapeutic agents used to target TfR1, antibodies stand out due to their remarkable specificity and affinity. Clinical trials are being conducted to evaluate the safety and efficacy of agents targeting TfR1 in cancer patients with promising results. These observations suggest that therapies targeting TfR1 as direct therapeutics or delivery conduits remain an attractive alternative for the treatment of cancers that overexpress the receptor., (Copyright © 2016 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.)

Salmonella enterica, a species of facultative anaerobic bacteria, has demonstrated success as a live-attenuated bacterial vector for vaccination. S. enterica has also demonstrated promise as a therapeutic agent against cancer. Pre-clinical and clinical trials have shown that S. enterica is localized in both solid and semi-solid tumors as well as in metastatic tumors. Moreover, S. enterica reduces resistance to treatment with other agents. In this review we present the novel therapeutic anti-cancer approaches that use S. enterica both for its ability as a delivery system for heterologous moieties against cancer and for its direct anti-cancer properties., (Copyright © 2014 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.)

Breast cancer remains the second leading cause of cancer death among women in the United States. Breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin(TM) family of anti-cancer drugs based on biodegradable and non-toxic polymalic acid (PMLA) was engineered for a multi-pronged attack on HER2/neu-positive breast cancer cells. An antibody-cytokine fusion protein consisting of the immunostimulatory cytokine interleukin-2 (IL-2) genetically fused to an antibody specific for human HER2/neu [anti-HER2/neu IgG3-(IL-2)] was covalently attached to the PMLA backbone to target HER2/neu expressing tumors and ensure the delivery of IL-2 to the tumor microenvironment. Antisense oligonucleotides (AON) were conjugated to the nanodrug to inhibit the expression of vascular tumor protein laminin-411 in order to block tumor angiogenesis. It is shown that the nanobioconjugate was capable of specifically binding human HER2/neu and retained the biological activity of IL-2. We also showed the uptake of the nanobioconjugate into HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. The nanobioconjugate exhibited marked anti-tumor activity manifested by significantly longer animal survival and significantly increased anti-HER2/neu immune response in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. The combination of laminin-411 AON and antibody-cytokine fusion protein on a single polymeric platform results in a new nanobioconjugate that can act against cancer cells through inhibition of tumor growth and angiogenesis and the orchestration of an immune response against the tumor. The present Polycefin(TM) variant may be a promising agent for treating HER2/neu expressing tumors and demonstrates the versatility of the Polycefin(TM) nanobioconjugate platform., (Copyright © 2013. Published by Elsevier B.V.)

Salmonella enterica is a facultative anaerobic bacteria, whose ability to colonize antigen-presenting cells (APCs) such as dendritic cells and macrophages, has allowed its successful use as an alive, attenuated bacterial vector for vaccination. Salmonella enterica elicits efficient cellular, humoral and mucosal immune responses, against heterologous antigens including viruses, parasites, other bacterial species and tumor-associated antigens, since it is capable of delivering these antigens to cells of the immune system. The extracellular expression of heterologous antigens on the surface of Salmonella enterica via its type I, III and V secretion systems, and their delivery into infected cells is essential for its stimulation of immune responses against these antigens. Moreover, Salmonella enterica is a promising therapeutic agent against cancer, as demonstrated by reports of pre-clinical and clinical studies indicating that, after systemic administration, Salmonella enterica preferentially localizes in solid tumors and metastases as compared to normal tissues. In this review, we focus on novel prophylactic and therapeutic anti-cancer approaches using Salmonella enterica as a delivery system of heterologous molecules with the aim of inhibiting tumor growth.

A number of antibodies have been developed that induce lethal iron deprivation (LID) by targeting the transferrin receptor 1 (TfR1/CD71) and either neutralizing transferrin (Tf) binding, blocking internalization of the receptor and/or inducing its degradation. We have developed recombinant antibodies targeting human TfR1 (ch128.1 and ch128.1Av), which induce receptor degradation and are cytotoxic to certain malignant B-cells. We now show that internalization of TfR1 bound to these antibodies can lead to its sequestration and degradation, as well as reduced Tf uptake, and the induction of a transcriptional response consistent with iron deprivation, which is mediated in part by downstream targets of p53. Cells resistant to these antibodies do not sequester and degrade TfR1 after internalization of the antibody/receptor complex, and accordingly maintain their ability to internalize Tf. These findings are expected to facilitate the rational design and clinical use of therapeutic agents targeting iron import via TfR1 in hematopoietic malignancies.

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, also known as CD71), which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fcγ receptors and the complement component C1q, suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition, in 2 disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant antitumor activity, including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies.

Conventional approaches for the detection of antibody dependent cell-mediated cytotoxicity (ADCC) activity rely on quantification of the release of traceable compounds from target cells or flow cytometry analysis of population-wide phenomena. We report a new method for the direct imaging and quantification of ADCC of cancer cells. The proposed method using imaging flow cytometry combines the statistical power of flow cytometry with the analytical advantages of cell imaging, providing a novel and more comprehensive perspective of effector/target cell interactions during ADCC events. With this method we can quantify and show in detail the morphological changes in target and effector cells, their apoptotic index, the physical interaction between effector and target cells, and a directional transfer of cytosolic contents from effector to target cells. As a model system we used the therapeutic anti-CD20 antibody rituximab to target CFSE labeled Ramos human Burkitt's lymphoma cells, to CMTPX-labeled human monocytic U-937 effector cells. We expect that similar studies using different effector and target cell populations may contribute to the pre-clinical evaluation of therapeutic antibodies and help to identify mechanisms that could be beneficial in the immunotherapy of cancer., (Copyright © 2011 Elsevier B.V. All rights reserved.)

DNA priming-protein boosting is a strategy used to establish strong immunity to a specific pathogen by the use of two different antigens through sequential delivery systems. In this work, two recombinant plasmids were used, one encoding for the dengue virus E protein, which is know to induce neutralizing antibodies (pcDNA 3.1/E), and the other encoding for the Dengue virus nonstructural protein 1 (pcDNA 3.1/NS1), as a source of B- and T-cell epitopes possibly involved in protective immunity. We showed that immunization of BALB/c mice with three priming doses of both plasmids pcDNA 3.1/E and/or pcDNA 3.1/NS1 were able to induce antibody responses to E protein with a single plasmid; in contrast to the antibody response to NS1 protein we observed an additive effect in terms of antibody response. Moreover, using a prime-boost protocol in which both plasmid constructs were co-administrated followed by a boost of homologous GST-E and GST-NS1 recombinant proteins, we observed an increased antibody response to NS1 and to E protein compared to animals vaccinated with the proteins or with dengue constructs alone. If neutralizing antibodies play an important role in dengue infection, antibodies generated with this regimen was also significantly better than the administration of the mix of proteins alone. These results suggest that NS1 and E proteins together could be considered in a design of subunit recombinant vaccines.