Studying the Humoral Response against SARS-CoV-2 in Cuban COVID-19 Recovered Patients.

Understanding the immune response generated by SARS-CoV-2 is critical for assessing efficient therapeutic protocols and gaining insights into the durability of protective immunity. The current work was aimed at studying the specific humoral responses against SARS-CoV-2 in Cuban COVID-19 convalescents. We developed suitable tools and methods based on ELISA methodology, for supporting this evaluation. Here, we describe the development of an ELISA for the quantification of anti-RBD IgG titers in a large number of samples and a similar test in the presence of NH4SCN as chaotropic agent for estimating the RBD specific antibody avidity. Additionally, a simple and rapid ELISA based on antibody-mediated blockage of the binding RBD-ACE2 was implemented for detecting, as a surrogate of conventional test, the levels of anti-RBD inhibitory antibodies in convalescent sera. In a cohort of 273 unvaccinated convalescents, we identified higher anti-RBD IgG titer (1 : 1,330, p < 0.0001) and higher levels of inhibitory antibodies blocking RBD-ACE2 binding (1 : 216, p < 0.05) among those who had recovered from severe illness. Our results suggest that disease severity, and not demographic features such as age, sex, and skin color, is the main determinant of the magnitude and neutralizing ability of the anti-RBD antibody response. An additional paired longitudinal assessment in 14 symptomatic convalescents revealed a decline in the antiviral antibody response and the persistence of neutralizing antibodies for at least 4 months after the onset of symptoms. Overall, SARS-CoV-2 infection elicits different levels of antibody response according to disease severity that declines over time and can be monitored using our homemade serological assays.

A cell-based ELISA as surrogate of virus neutralization assay for RBD SARS-CoV-2 specific antibodies.

SARS-CoV-2, the cause of the COVID-19 pandemic, has provoked a global crisis and death of millions of people. Several serological assays to determine the quality of the immune response against SARS-CoV-2 and the efficacy of vaccines have been developed, among them the gold standard conventional virus neutralization assays. However, these tests are time consuming, require biosafety level 3 (BSL3), and are low throughput and expensive. This has motivated the development of alternative methods, including molecular inhibition assays. Herein, we present a safe cell-based ELISA-virus neutralization test (cbE-VNT) as a surrogate for the conventional viral neutralization assays that detects the inhibition of SARS-CoV-2 RBD binding to ACE2-bearing cells independently of species. Our test shows a very good correlation with the conventional and molecular neutralization assays and achieves 100% specificity and 95% sensitivity. cbE-VNT is cost-effective, fast and enables a large-scale serological evaluation that can be performed in a BSL2 laboratory, allowing its use in pre-clinical and clinical investigations.

Bacitracin is a non-competitive inhibitor of porcine M1 family neutral and glutamyl aminopeptidases.

Membrane alanyl and glutamyl aminopeptidases (APN and APA, respectively) are established targets for the development of biomedical tools in human pathologies. APN overexpression correlates with the progression of tumours, including melanoma. Bacitracin, widely used as a topical antibiotic, inhibits subtilisin-like serine peptidases and disulphide isomerases. In the present contribution, we demonstrate that bacitracin is a non-competitive α = 1 and α < 1 inhibitor of porcine kidney APN and APA, respectively, with Ki values in the micromolar range. To test a potential application of this result, we assayed the effect of bacitracin on murine melanoma MB16F10 cell line viability. We demonstrated the cell line expresses an APN-like activity inhibited by bacitracin and bestatin. Additionally, we identified a cytotoxic effect of bacitracin. Further experiments are required to understand in depth the mechanisms of action of bacitracin on melanoma cells. They will clarify the therapeutic potential of bacitracin for melanoma treatment.

Bestatin and bacitracin inhibit porcine kidney cortex dipeptidyl peptidase IV activity and reduce human melanoma MeWo cell viability.

Bestatin and bacitracin are inhibitors of metallo aminopeptidases and bacterial proteases. However, their effects on other human peptidases, like dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) are not established. Inhibitors of DPP-IV activity are used for treating type 2 diabetes mellitus, cancers and immune system diseases. Bacitracin and bestatin inhibited porcine membrane-bound DPP-IV (pDPP-IV) activity. Mechanisms were different, i.e. non-competitive with α > 1 (α = 3.9) and Ki value of 75 µM for bestatin, and competitive with Ki value of 630 µM for bacitracin. The binding mode in the tertiary complex enzyme:substrate:bestatin suggested the structural basis of the inhibitory effect and that bestatin is potentially selective for DPP-IV, ineffective vs. S9 family members dipeptidyl peptidase 8/9 and fibroblast activation protein. In the human melanoma MeWo cell line, bestatin and bacitracin inhibited aminopeptidase N (APN) and DPP-IV activities, reduced cell viability and increased DNA fragmentation, suggesting induction of apoptosis. Since bacitracin and bestatin are already marketed drugs, studying in depth the molecular mechanisms underlying their effects on melanoma cells is warranted. Additionally, bestatin emerges as a new lead compound for the development of DPP-IV inhibitors, and a promising dual APN/DPP-IV inhibitor for the treatment of pathologies in which both enzymes are upregulated.

Safety and Immunogenicity of a Human Epidermal Growth Factor Receptor 1 (HER1)-Based Vaccine in Prostate Castration-Resistant Carcinoma Patients: A Dose-Escalation Phase I Study Trial.

Metastatic castration-resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. Activation of the human epidermal growth factor receptor 1 (HER1) in prostate cancer contributes to metastatic progression as well as to disease relapse. Here, we determined the toxicity and immunogenicity of a HER1-based cancer vaccine in CRPC patients included in a phase I clinical trial. CRPC patients (n = 24) were intramuscularly vaccinated with HER1 vaccine consisting of the extracellular domain of HER1 molecule (ECD) and very small size proteoliposome from Neisseria meningitidis (VSSP) and Montanide ISA-51 VG as adjuvants. Patients were included in five groups according to the vaccine dose (100, 200, 400, 600, and 800 µg). The primary endpoints were safety and immunogenicity. The anti-HER1 antibodies were measured by an ELISA, the recognition of an HER1 positive tumor cell line and the inhibition of HER1 phosphorylation by sera were determined by flow cytometry and western blot analysis, respectively. The HER1-specific T cell response was assessed by determination of IFN-γ-producing T cells using ELISpot assay. The vaccine was well tolerated. No grade III or IV adverse events were reported. High titers of anti-HER1 antibodies were observed in most of the evaluated patients. There were no significant differences regarding the geometric means of the anti-HER1 titers among the dose groups except the group of 100 µg in which antibody titers were significantly lower. A Th1-type IgG subclasses pattern was predominant in most patients. Only patients receiving the higher doses of vaccine showed significant tumor cell recognition and HER1 phosphorylation inhibition by hyperimmune sera. Forty two percent of the patients showed a specific T cell response against HER1 peptides pool in post-treatment samples. There was a trend toward survival benefit in those patients showing high anti-HER1 specific antibody titers and a significant association between cellular immune response and clinical outcome.

Comparative proteomic analysis of growth hormone secretagogue A233 treatment of murine macrophage cells J774A.2 indicates it has a role in antiviral innate response.

BACKGROUND: Growth hormone secretagogues (GHS), among other factors, regulate the release of GH. The biological activity of the secretagogue peptide A233 as a promoter of growth and innate immunity in teleost fish has previously been demonstrated, but its role in the immune system of mammals is not well understood. METHODS: The effect of the peptide was investigated in J774A.2 macrophage cells using a comparative proteomics approach after 6 and 12 h of peptide stimulation. RESULTS: The functional analysis of differentially modulated proteins showed that A233 peptide treatment appears to promote activation and ROS-dependent cytotoxic functions in macrophages and enhanced expression of antiviral protein complexes such as MAVS. In accordance with this hypothesis, we found that A233 treatment enhanced superoxide anion production and the IFN-γ level in J774A.2 cells and mouse splenocytes, respectively, and reduced viral load in a dengue virus mouse model of infection. CONCLUSIONS: The growth hormone secretagogue A233 peptide promotes activation of ROS-dependent cytotoxic functions and exerts immunomodulatory effects that enable an antiviral state in a dengue virus mouse model. GENERAL SIGNIFICANCE: The increase of IFN-γ level and the differential modulation of antiviral proteins by the A233 peptide suggest that the molecule could activate an innate immune response with a possible further impact in the treatment of acute and chronic diseases.

Effects of an epidermal growth factor receptor-based cancer vaccine on wound healing and inflammation processes in murine experimental models.

Anti-epidermal growth factor receptor (EGFR) therapies have been proven clinically effective for a variety of epithelial tumours. Vaccination of mice with the extracellular domain (ECD) of autologous EGFR overcomes the tolerance to self-EGFR and has antimetastatic effect on EGFR+ tumour. Because EGF/EGFR-signalling plays an important role in the inflammation stage of wound healing, the main objective of this study was to explore the possible role of murine (m) EGFR-ECD vaccine in the croton-oil-induced ear oedema and wound healing process in mice as autologous experimental models, mimicking the possible post-surgical wound complication in patients treated with human EGFR-ECD/VSSP vaccine. Mice were intramuscularly immunised four times; biweekly with the mEGFR-ECD/VSSP/Mont. Seven days later, an 8 mm diameter, full-thickness skin wound was created on the back of each animal. Immunisation induced a strong specific humoral response against the mEGFR-ECD protein and a DTH dose-response curve but interestingly, animals treated with mEGFR-ECD/VSSP/Mont had similar inflammatory and healing speed responses compared to control ones. These data suggest that application of mEGFR-ECD/VSSP vaccine as a therapeutic approach in cancer patients could not elicit a poor healing process after surgery.

Challenges and opportunities for cancer vaccines in the current NSCLC clinical scenario.

This review is aimed to focus on NSCLC as an emerging and promising model for active immunotherapy and the challenges for its inclusion in the current clinical scenario. Cancer vaccines for NSCLC have been focused as a therapeutic option based on the identification of a tumor hallmark and the active immunization with the related molecules that triggers cellular and/or humoral responses that consequently destroy or delay the rate of malignant progression. This therapeutic intervention in an established disease state has been aimed to impact into prolonging patient´s survival with ethically accepted quality of life. Understanding of relationship between structure and function in cancer vaccines is essential to interpret their opportunities to impact into prolonging survival and increasing quality of life in cancer patients. It is widely accepted that the failure of the cancer vaccines in the NSCLC scenario is related with its introduction in the advanced disease stages and poor performance status of the patients due to the combination of the tumor induced immunosuppression with the immune senescence. Despite first, second and emerging third line of onco-specific treatments the life expectancy for NSCLC patients diagnosed at advanced stages is surrounding the 12 months of median survival and in facts the today real circumstances are extremely demanding for the success inclusion of cancer vaccines as therapeutic choice in the clinical scenario. The kinetics of the active immunizations encompasses a sequential cascade of clinical endpoints: starting by the activation of the immune system, followed by the antitumor response and finalizing with the consequential impact on patients' overall survival. Today this cascade of clinical endpoints is the backbone for active immunization assessment and moreover the concept of cancer vaccines, applied in the NSCLC setting, is just evolving as a complex therapeutic strategy, in which the opportunities for cancer vaccines start from the selection of the target cancer hallmark, followed by the vaccine formulation and its platforms for immune potentiating, also cover the successful insertion in the standard of care, the chronic administration beyond progression disease, the personalization based on predictors of response and the potential combination with other targeted therapies.

Linking oncogenesis and immune system evasion in acquired resistance to EGFR-targeting antibodies: Lessons from a preclinical model.

Searching for biomarkers that associated with the acquired resistance of malignant cells to epidermal growth factor receptor (EGFR)-targeting monoclonal antibodies is crucial to improve the clinical benefits of these therapeutic agents. We have recently demonstrated that molecular alterations in both oncogenic and immunological pathways may be responsible for such an insensitivity. Our findings suggest that a combination of targeted anticancer agents and immunomodulatory drugs may be useful for overcoming the acquired resistance of cancer cells to EGFR-specific monoclonal antibodies.

Comparative in vitro and experimental in vivo studies of the anti-epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants.

A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.

Induction of immunogenic apoptosis by blockade of epidermal growth factor receptor activation with a specific antibody.

Despite promising results in the use of anti-epidermal growth factor receptor (EGFR) Abs for cancer therapy, several issues remain to be addressed. An increasing emphasis is being placed on immune effector mechanisms. It has become clear for other Abs directed to tumor targets that their effects involve the adaptive immunity, mainly by the contribution of Fc region-mediated mechanisms. Given the relevance of EGFR signaling for tumor biology, we wonder whether the oncogene inhibition could contribute to Ab-induced vaccine effect. In a mouse model in which 7A7 (an anti-murine EGFR Ab) and AG1478 (an EGFR-tyrosine kinase inhibitor) displayed potent antimetastatic activities, depletion experiments revealed that only in the case of the Ab, the effect was dependent on CD4(+) and CD8(+) T cells. Correspondingly, 7A7 administration elicited a remarkable tumor-specific CTL response in hosts. Importantly, experiments using 7A7 F(ab')(2) suggested that in vivo Ab-mediated EGFR blockade may play an important role in the linkage with adaptive immunity. Addressing the possible mechanism involved in this effect, we found quantitative and qualitative differences between 7A7 and AG1478-induced apoptosis. EGFR blocking by 7A7 not only prompted a higher proapoptotic effect on tumor metastases compared with AG1478, but also was able to induce apoptosis with immunogenic potential in an Fc-independent manner. As expected, 7A7 but not AG1478 stimulated exposure of danger signals on tumor cells. Subcutaneous injection of 7A7-treated tumor cells induced an antitumor immune response. This is the first report, to our knowledge, of a tumor-specific CTL response generated by Ab-mediated EGFR inhibition, suggesting an important contribution of immunogenic apoptosis to this effect.

Purification process development for HER1 extracellular domain as a potential therapeutic vaccine.

HER1 is a tumor associated antigen emerging as an attractive target for cancer therapy. In the present study we demonstrated for first time that HER1 extracellular domain can be purified by a downstream process at pilot scale based on immunoaffinity chromatography from bioreactor supernatant of HEK 293 transfectomes. Filtered supernatant was applied to CNBr-activated Sepharose CL-4B with monoclonal antibody anti-human EGF immobilized, followed by three additional chromatographic polishing steps. HER1 extracellular domain was obtained with high purity (>95%), low DNA content, and biological activity.

T cells are crucial for the anti-metastatic effect of anti-epidermal growth factor receptor antibodies.

Experimental evidences supporting the epidermal growth factor receptor (EGFR) as an important molecule for tumor metastasis had been accumulated. Currently, anti-EGFR monoclonal antibodies (mAbs) constitute a promising approach for the treatment of patients with metastatic tumors. However, the mechanisms associated with the potent anti-metastatic effect of these mAbs have not been completely elucidated due to the lack of appropriate syngeneic preclinical models. In this paper, we have investigated the effects of 7A7, an antibody specific to murine EGFR, on the metastatic properties of D122 murine lung carcinoma. 7A7 mAb significantly impaired metastatic spread of D122 cells in C57BL/6 mice by direct anti-proliferative and pro-apoptotic effects on tumor metastasis. 7A7 mAb capacity to inhibit EGFR activation on D122 cells could contribute to its anti-metastatic effect. In addition, 7A7 mAb was able to induce in vitro antibody-dependent cell-mediated cytotoxicity on D122 cells. Interestingly, 7A7 mAb treatment increased the number of natural killer cells, T lymphocytes and dendritic cells infiltrating the metastatic sites. More strikingly, depletion of CD8(+) and CD4(+) T cells in vivo completely abrogated the 7A7 mAb anti-metastatic activity whereas function of natural killer cells was irrelevant. This study supports an in vivo role for T cell response in the mechanism of action of anti-EGFR mAbs, suggesting the induction of an adjuvant effect.

7A7 MAb: a new tool for the pre-clinical evaluation of EGFR-based therapies.

The epidermal growth factor receptor (EGFR) is highly expressed in many types of epithelial tumors. EGFR overexpression has been associated with an advanced stage of the disease, with resistance to standard therapies, and, for certain tumors, with poor patient prognosis. As a result, EGFR has been considered a meaningful target in anti-tumor strategies. Active and passive immunotherapies blocking EGFR and its ligands have been explored. But for successful pre-clinical evaluation of these approaches, well-established murine tumor models are not available and highly desirable. We described, for the first time, the generation and characterization of an anti-murine EGFR extracellular domain monoclonal antibody (7A7 MAb) (IgG1). 7A7 was generated by immunization of Balb/c mice with the recombinant extracellular domain of murine EGFR (rECD-mEGFR). 7A7 recognized an epitope present in the amino acidic core of the antigen and is cross-reactive with the human EGFR. Interestingly, this MAb was able to specifically bind EGFR at the cell surface, allowing the assessment of its differential expression in a panel of murine cells. Noteworthy, in a preliminary immunohistochemical study with 7A7 MAb, recognition of Balb/c mice skin sections and EGFR-positive tumors were observed. We concluded that 7A7 MAb is a valuable tool for EGFR-based therapeutic pre-clinical studies.