BioHabana 2022: Preventive and Immunotherapeutic Strategies Against COVID-19 and Cancer in Cuba.

The convergence of life sciences with neurosciences, nanotechnology, data management, and engineering has caused a technological diversification of the biotechnology, pharmaceutical, and medical technology industries, including the phenomenon of digital transformation, which has given rise to the so-called Fourth Industrial Revolution (Industry 4.0). Confronting the COVID-19 pandemic revealed the outstanding response capacity of the scientific community and the biopharmaceutical industry, based on a multidisciplinary and interinstitutional approach that has achieved an unprecedented integration in the history of biomedical science. Cuba, a small country, with scarce material resources, has had remarkable success in controlling the disease, which also highlights the impact of social factors. This report presents a summary of the most relevant presentations of selected topics during the scientific meeting, "BioHabana 2022: Cancer Immunotherapy and the COVID-19 Pandemic," which was held in Havana Cuba in April 2022.

A co-formulation of interferons alpha2b and gamma distinctively targets cell cycle in the glioblastoma-derived cell line U-87MG.

BACKGROUND: HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model. METHODS: Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry. RESULTS: The three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG. CONCLUSIONS: We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.

Gene expression profiling unveils the temporal dynamics of CIGB-300-regulated transcriptome in AML cell lines.

BACKGROUND: Protein kinase CK2 activity is implicated in the pathogenesis of various hematological malignancies like Acute Myeloid Leukemia (AML) that remains challenging concerning treatment. This kinase has emerged as an attractive molecular target in therapeutic. Antitumoral peptide CIGB-300 blocks CK2 phospho-acceptor sites on their substrates but it also binds to CK2α catalytic subunit. Previous proteomic and phosphoproteomic experiments showed molecular and cellular processes with relevance for the peptide action in diverse AML backgrounds but earlier transcriptional level events might also support the CIGB-300 anti-leukemic effect. Here we used a Clariom S HT assay for gene expression profiling to study the molecular events supporting the anti-leukemic effect of CIGB-300 peptide on HL-60 and OCI-AML3 cell lines. RESULTS: We found 183 and 802 genes appeared significantly modulated in HL-60 cells at 30 min and 3 h of incubation with CIGB-300 for p < 0.01 and FC > = │1.5│, respectively; while 221 and 332 genes appeared modulated in OCI-AML3 cells. Importantly, functional enrichment analysis evidenced that genes and transcription factors related to apoptosis, cell cycle, leukocyte differentiation, signaling by cytokines/interleukins, and NF-kB, TNF signaling pathways were significantly represented in AML cells transcriptomic profiles. The influence of CIGB-300 on these biological processes and pathways is dependent on the cellular background, in the first place, and treatment duration. Of note, the impact of the peptide on NF-kB signaling was corroborated by the quantification of selected NF-kB target genes, as well as the measurement of p50 binding activity and soluble TNF-α induction. Quantification of CSF1/M-CSF and CDKN1A/P21 by qPCR supports peptide effects on differentiation and cell cycle. CONCLUSIONS: We explored for the first time the temporal dynamics of the gene expression profile regulated by CIGB-300 which, along with the antiproliferative mechanism, can stimulate immune responses by increasing immunomodulatory cytokines. We provided fresh molecular clues concerning the antiproliferative effect of CIGB-300 in two relevant AML backgrounds.

Proteomics and Phospho-Proteomics Profiling of the Co-Formulation of Type I and II Interferons, HeberFERON, in the Glioblastoma-Derived Cell Line U-87 MG.

HeberFERON, a co-formulation of Interferon (IFN)-α2b and IFN-γ, has effects on skin cancer and other solid tumors. It has antiproliferative effects over glioblastoma multiform (GBM) clones and cultured cell lines, including U-87 MG. Here, we report the first label-free quantitative proteomic and phospho-proteomic analyses to evaluate changes induced by HeberFERON after 72 h incubation of U-87 MG that can explain the effect on cellular proliferation. LC-MS/MS, functional enrichment and networking analysis were performed. We identified 7627 proteins; 122 and 211 were down- and up-regulated by HeberFERON (fold change > 2; p < 0.05), respectively. We identified 23,549 peptides (5692 proteins) and 8900 phospho-peptides; 523 of these phospho-peptides (359 proteins) were differentially modified. Proteomic enrichment showed IFN signaling and its control, direct and indirect antiviral mechanisms were the main modulated processes. Phospho-proteome enrichment displayed the cell cycle as one of the most commonly targeted events together with cytoskeleton organization; translation/RNA splicing, autophagy and DNA repair, as represented biological processes. There is a high interconnection of phosphoproteins in a molecular network; mTOR occupies a centric hub with interactions with translation machinery, cytoskeleton and autophagy components. Novel phosphosites and others with unknown biological functionality in key players in the aforementioned processes were regulated by HeberFERON and involved CDK and ERK kinases. These findings open new experimental hypotheses regarding HeberFERON action. The results obtained contribute to a better understanding of HeberFERON effector mechanisms in the context of GBM treatment.

Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection.

Coronaviruses constitute a global threat to the human population; therefore, effective pan-coronavirus antiviral drugs are required to tackle future re-emerging virus outbreaks. Protein kinase CK2 has been suggested as a promising therapeutic target in COVID-19 owing to the in vitro antiviral activity observed after both pharmacologic and genetic inhibition of the enzyme. Here, we explored the putative antiviral effect of the anti-CK2 peptide CIGB-325 on bovine coronavirus (BCoV) infection using different in vitro viral infected cell-based assays. The impact of the peptide on viral mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. Finally, pull-down experiments followed by Western blot and/or mass spectrometry analysis were performed to identify CIGB-325-interacting proteins. We found that CIGB-325 inhibited both the cytopathic effect and the number of plaque-forming units. Accordingly, intracellular viral protein levels were clearly reduced after treatment of BCoV-infected cells, with CIGB-325 determined by immunocytochemistry. Pull-down assay data revealed the physical interaction of CIGB-325 with viral nucleocapsid (N) protein and a group of bona fide CK2 cellular substrates. Our findings evidence in vitro antiviral activity of CIGB-325 against bovine coronavirus as well as some molecular clues that might support such effect. Altogether, data provided here strengthen the rationale of inhibiting CK2 to treat betacoronavirus infections.

Reliability of CNEURO hyssops for sample collection in the SARS-CoV-2 diagnosis / Fiabilidad de los hisopos CNEURO para la recogida de muestras en el diagnóstico del SARS-CoV-2

ABSTRACT Introduction: The detection of SARS-CoV-2 genetic material from nasopharyngeal swab samples by RT-PCR is the most specific and sensitive way to test suspected cases. However, factors such as the sampling process, the type of hyssop used, and the anatomical area from which the sample is collected can distort the result and cause false negatives. Objective: To evaluate the reliability of CNUERO hyssops for sample collection for the SARS-CoV-2 diagnosis versus IMPROSWAB hyssops. Material and Methods: To study the reliability of hyssops developed in Cuba for swabbing for the COVID-19 diagnosis by comparing them to other hyssops successfully used for this task, 2 swabbing samples were obtained from each patient (136). One of these two samples was taken using the hyssops made in Cuba, while the other was taken using another hyssop imported from Germany. The positive detections obtained with the use of both hyssops were compared using the Fisher's exact test. The result of the detection of each hyssop was evaluated and compared using the ROC curve. Results: The use of CNEURO hyssops allowed the detection of 45 out of 59 positive cases, while IMPROSAWAB hyssops detected 52 out of 59 true positive cases. There were no significant differences between positive cases detected with the use of each hyssop. The sensitivity of sample detection using CNEURO hyssops was 76,3 % while the one using IMPROSWAB hyssops was 88,1 %. Hence, there are no significant differences in the detection of cases using these two hyssops. Conclusion: CNEURO hyssops are safe and reliable to be used to take nasopharyngeal samples from COVID-19 patients.

RESUMEN Introducción: La detección de material genético del SARS-CoV-2 a partir de muestras de hisopos nasofaríngeos mediante RT-PCR es la forma más específica y sensible de analizar los casos sospechosos. Sin embargo, factores como el proceso de toma de muestra, el tipo de hisopo y el área anatómica de la que se extrae la muestra, pueden distorsionar el resultado y provocar falsos negativos. Objetivo: Evaluar la confiabilidad de hisopos CNUERO para la recolección de muestras en el diagnóstico de SARS-CoV-2 versus hisopos IMPROSWAB. Material y Métodos: Se obtuvieron 2 muestras de exudado de cada paciente (136). Una de estas dos muestras se tomó con hisopos CNEURO, mientras que la otra se tomó con el hisopo IMPROSWAB. Las detecciones positivas entre ambos hisopos se compararon mediante la prueba exacta de Fisher. El resultado de la detección de cada hisopo se evaluó y comparó utilizando la curva ROC. Resultados: El uso de hisopos CNEURO permitió detectar 45 de 59 casos positivos, mientras que los hisopos IMPROSAWAB detectaron 52 de 59 casos verdaderos positivos. Se detectaron diferencias no significativas entre los casos positivos detectados entre hisopos. La sensibilidad de detección de muestras utilizando hisopos CNEURO fue del 76,3 % y del 88,1 % cuando se utilizaron hisopos IMPROSWAB. Por tanto, no se detectaron diferencias significativas en la detección de casos utilizando estos dos hisopos. Conclusión: Los hisopos CNEURO son seguros y fiables para su uso en la toma de muestras nasofaríngeas de pacientes con COVID-19.

CIGB-300 Anticancer Peptide Differentially Interacts with CK2 Subunits and Regulates Specific Signaling Mediators in a Highly Sensitive Large Cell Lung Carcinoma Cell Model.

Large cell lung carcinoma (LCLC) is one form of NSCLC that spreads more aggressively than some other forms, and it represents an unmet medical need. Here, we investigated for the first time the effect of the anti-CK2 CIGB-300 peptide in NCI-H460 cells as an LCLC model. NCI-H460 cells were highly sensitive toward CIGB-300 cytotoxicity, reaching a peak of apoptosis at 6 h. Moreover, CIGB-300 slightly impaired the cell cycle of NCI-H460 cells. The CIGB-300 interactomics profile revealed in more than 300 proteins that many of them participated in biological processes relevant in cancer. Interrogation of the CK2 subunits targeting by CIGB-300 indicated the higher binding of the peptide to the CK2α' catalytic subunit by in vivo pull-down assays plus immunoblotting analysis and confocal microscopy. The down-regulation of both phosphorylation and protein levels of the ribonuclear protein S6 (RPS6) was observed 48 h post treatment. Altogether, we have found that NCI-H460 cells are the most CIGB-300-sensitive solid tumor cell line described so far, and also, the findings we provide here uncover novel features linked to CK2 targeting by the CIGB-300 anticancer peptide.

Targeting of Protein Kinase CK2 in Acute Myeloid Leukemia Cells Using the Clinical-Grade Synthetic-Peptide CIGB-300.

Protein kinase CK2 has emerged as an attractive therapeutic target in acute myeloid leukemia (AML), an advent that becomes particularly relevant since the treatment of this hematological neoplasia remains challenging. Here we explored for the first time the effect of the clinical-grade peptide-based CK2 inhibitor CIGB-300 on AML cells proliferation and viability. CIGB-300 internalization and subcellular distribution were also studied, and the role of B23/nucleophosmin 1 (NPM1), a major target for the peptide in solid tumors, was addressed by knock-down in model cell lines. Finally, pull-down experiments and phosphoproteomic analysis were performed to study CIGB-interacting proteins and identify the array of CK2 substrates differentially modulated after treatment with the peptide. Importantly, CIGB-300 elicited a potent anti-proliferative and proapoptotic effect in AML cells, with more than 80% of peptide transduced cells within three minutes. Unlike solid tumor cells, NPM1 did not appear to be a major target for CIGB-300 in AML cells. However, in vivo pull-down experiments and phosphoproteomic analysis evidenced that CIGB-300 targeted the CK2α catalytic subunit, different ribosomal proteins, and inhibited the phosphorylation of a common CK2 substrates array among both AML backgrounds. Remarkably, our results not only provide cellular and molecular insights unveiling the complexity of the CIGB-300 anti-leukemic effect in AML cells but also reinforce the rationale behind the pharmacologic blockade of protein kinase CK2 for AML-targeted therapy.

Comparison between nasopharyngeal swabs and saliva as reliable specimens for the diagnosis of SARS-CoV-2 / Comparación de la confiabilidad del diagnóstico del SARS-CoV-2 entre muestras de hisopado y saliva

Introduction: The SARS-CoV-2 virus is a positive-strand RNA virus. The virus can also be detected in many different specimens as throat swabs, nasal swabs, sputum, saliva, blood, etc. Objective: The aim of this paper is to compare the reliability of different types of specimen collection, saliva and swabs samples for the detection of SARS-CoV-2. Material and Methods: A sample of 22 COVID-19 positive patients was selected. Paired samples from saliva, nasopharyngeal, oropharyngeal and nasopharyngeal + oropharyngeal swabs were collected on the 7th day after diagnosis. The hyssops and medium employed was IMPROSWAB and IMPROVIRAL NAT Medium, Germany. The sample evaluation was conducted through RT-PCR. The results were compared using Fisher's exact test and ROC curve. The gold standard proposed in this paper was the nasopharyngeal + oropharyngeal swabs specimen. Results: The gold standard method detected 10 true positive cases, of which oropharyngeal swabs, nasopharyngeal swabs and saliva only detected three positive cases. Significant differences (Fisher's exact test p = 0.003) were detected in the comparison between saliva and the gold standart proposed. The ROC curve analysis showed that saliva had an area under the curve of 0.650, with a 30 percent of sensibility. However, the nasopharyngeal and nasopharyngeal + oropharyngeal samples had an area under curve of 0.950 and 1.000, respectively, with a sensibility of 90 percent and 100 percent, respectively. Conclusion: Saliva samples are not a reliable specimen for SARS-CoV-2 RNA detection. In turn, the most reliable specimens are nasopharyngeal and nasopharyngeal + oropharyngeal samples collected by swabbing(AU)

Introducción: El SARS-CoV-2 es un virus ARN positivo. Este virus puede ser detectado en diferentes tipos de secreción como hisopada bucal, nasal, esputo, saliva, sangre, etc. Objetivo: El objetivo de este estudio es comparar la confiabilidad de diferentes tipos de muestras, saliva y exudado, en la detección de SARS-CoV-2. Material y Métodos: Una muestra de 22 pacientes con diagnóstico de Covid-19 fue estudiada. Se tomaron muestras pareadas de saliva y exudado nasofaríngeo y orofaríngeo en cada paciente. Se emplearon los hisopos y medios de la firma alemana IMPROVE®. Los resultados de las determinaciones por RT-PCR se compararon mediante test de Fisher (test de la probabilidad exacta de Fisher) y cada sets de muestras fue evaluada individualmente y luego comparadas por curvas ROC. El estándar de oro propuesto fue el doble hisopado nasofaríngeo/orofaríngeo. Resultados: El método de oro propuesto detectó 10 casos positivos. La coincidencia de detección entre todos los sets de muestras fue de 3 casos (30 por ciento). Se obtuvieron diferencias significativas (Fisher p = 0.003) en la comparación de los casos detectados en saliva vs el estándar de oro. El análisis de curvas ROC mostró un área bajo la curva de 0.650 (30 por ciento de sensibilidad) para la saliva. En el caso del hisopado nasofaríngeo y el estándar de oro mostraron un área bajo la curva de 0.95 y 1.00, respectivamente, con una sensibilidad del 90 (AU) por ciento y 100 por ciento, respectivamente. Conclusiones: La saliva no es una muestra confiable para la detección de SARS-CoV-2. La muestra más confiable para el diagnóstico fue el hisopado nasofaríngeo y el doble hisopado(AU)

A co-formulation of interferons type I and II enhances temozolomide response in glioblastoma with unmethylated MGMT promoter status.

Temozolomide (TMZ) is a chemotherapeutic used for the treatment of glioblastoma. The MGMT repair enzyme (O'-(6)-methyl guanine-DNA-methyltransferase) promoter methylation is a predictive biomarker to TMZ response; interferons (IFNs) type I can downregulate MGMT expression improving survival in patients with unmethylated MGMT promoter. HeberFERON is a co-formulation of IFNs type I and II with higher antiproliferative effect over glioblastoma cell lines than individual IFNs. We investigated the proliferative response of patient-derived glioblastoma cultures to HeberFERON and its combination with TMZ in relation to MGMT promoter methylation and the regulation of MGMT transcript after HeberFERON treatment. Eleven glioblastoma-derived cultures, molecularly classified according to TCGA and MGMT promoter methylation, were assayed for proliferation inhibition with HeberFERON at low doses (1-25 IU/mL) [alone or combined with TMZ] or at higher doses (50-200 IU/mL) using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Eight cultures were further treated with 100 IU/mL of HeberFERON for 72 h, total RNA purified (Qiagen) and converted to cDNA (Superscript III kit, Invitrogen) as quantitative PCR templates. Changes of MGMT&P53 transcripts level were monitored. Response of cultures to HeberFERON is variable, dose-dependent and apparently independent from TCGA classification and MGMT methylation status, based on the eight Classical cultures data. When combining HeberFERON with TMZ there was an increase in cell death for cultures, 2/4 with methylated and 5/5 with unmethylated MGMT promoter. In two out five cultures with unmethylated MGMT status, we observed a decrease of MGMT gene levels and an increase in P53 encoding gene levels. HeberFERON and TMZ combination should be further assayed in glioblastoma, mainly for those with unmethylated MGMT promoter.

Regulation by IFN-α/IFN-γ co-formulation (HerberPAG®) of genes involved in interferon-STAT-pathways and apoptosis in U87MG.

Interferons (IFNs) are proteins of the family of cytokines. Their antiproliferative function has been taken into account for several clinical therapies against malignant diseases. In this family, IFNs α and γ have demonstrated the highest antitumor effects. HerberPAG® is a new co-formulation with IFNs, α2b and γ. It has been obtained to increase the antiproliferative effect of individual IFNs and decrease their associated toxicity. Glioblastoma multiforme (GBM) is the most common primary brain tumor and one of the most deadly forms of cancer. The objective of the present work is to obtain insights into the regulation of Interferon-STAT-pathways and apoptosis in U87MG, at the transcriptional level. As a pharmacogenomic strategy we quantified mRNAs levels in vitro by quantitative PCR, using the cell line U87MG as a model. Some of the genes involved in the first steps of IFNs signaling pathways (stat1 and stat3) and apoptosis events (tp53, bax, bcl-2, bad, caspase3 (casp3), caspase8 (casp8) and caspase9 (casp9)) were studied. The detected mRNAs expression pattern for stat1and stat3 indicates a higher tumor suppressor activity of HerberPAG® compared to individuals IFNs. The up-regulation of tp53, bax, bad, casp3, casp8 and casp9 genes and the down regulation of bcl-2 gen, after the treatment with HerberPAG® show a pro-apoptotic function. HerberPAG® gene-induced profile shows an advantage in relation to IFN α2b and γ with a higher stat1 expression and a downregulation of bcl-2 which increases bax:bcl-2 ratio. The regulation of genes involved in IFN-STAT-pathways and apoptosis may be the first evidences to explain the increased antiproliferative properties of this co-formulation.

Selection of reference genes for use in quantitative reverse transcription PCR assays when using interferons in U87MG.

Relative gene quantification by quantitative reverse transcription PCR (qRT-PCR) is an accurate technique only when a correct normalization strategy is carried out. Some of the most commonly genes used as reference have demonstrated variation after interferon (IFN) treatments. In this work we evaluated the suitability of seven reference genes (RGs) [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), ß-2Microglobulin (B2M), ribosomal RNA subunits 18S and 28S, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) and the RNA helicase (DDX5)] for use in qRT-PCR assays in the glioblastoma-derived cell line U87MG treated with IFNα, IFNγ or a co-formulated combination of both IFNs (HeberPAG); untreated cell lines were included as control. Data was analyzed using geNorm and NormFinder softwares. The expression stability of the seven RGs decreased in order of DDX5/GAPDH/HMBS, 18S rRNA, YWHAZ, 28S rRNA and B2M. qRT-PCR analyses demonstrated that DDX5, GAPDH and HMBS were among the best stably expressed markers under all conditions. Both, geNorm and NormFinder, analyses proposed same RGs as the least variables. Evaluation of the expression levels of two target genes utilizing different endogenous controls, using REST-MCS software, revealed that the normalization method applied might introduce errors in the estimation of relative quantities. We concluded that when qRT-PCR is designed for studies of gene expression in U87MG cell lines treated with IFNs type I and II or their combinations, the use of all three GAPDH, HMBS and DDX5 (or their combinations in pairs) as RGs for data normalizations is recommended.

Pharmacokinetic and pharmacodynamic characterization of a new formulation containing synergistic proportions of interferons alpha-2b and gamma (HeberPAG) in patients with mycosis fungoides: an open-label trial.

BACKGROUND: The synergistic combination of interferon (IFN) alpha-2b and IFN gamma results in more potent in vitro biological effects mediated by both IFNs. The aim of this investigation was to evaluate by first time the pharmacokinetics and pharmacodynamics of this combination in patients with mycosis fungoides. METHODS: An exploratory, prospective, open-label clinical trial was conducted. Twelve patients, both genders, 18 to 75 years-old, with mycosis fungoides at stages IB to III, were eligible for the study. All of them received intramuscularly a single high dose (23 × 10(6) IU) of a novel synergistic IFN mixture (HeberPAG) for pharmacokinetic and pharmacodynamic studies. Serum IFN alpha-2b and IFN gamma concentrations were measured during 96 hours by commercial enzyme immunoassays (EIA) specific for each IFN. Other blood IFN-inducible markers and laboratory variables were used as pharmacodynamics and safety criteria. RESULTS: The pharmacokinetic evaluation by EIA yielded a similar pattern for both IFNs that are also in agreement with the well-known described profiles for these molecules when these are administered separately. The average values for main parameters were: Cmax: 263 and 9.3 pg/mL; Tmax: 9.5 and 6.9 h; AUC: 4483 and 87.5 pg.h/mL, half-life (t(1/2)): 4.9 and 13.4 h; mean residence time (MRT): 13.9 and 13.5 h, for serum IFN alpha-2b and IFN gamma, respectively. The pharmacodynamic variables were strongly stimulated by simultaneous administration of both IFNs: serum neopterin and beta-2 microglobulin levels (ß2M), and stimulation of 2'-5' oligoadenylate synthetase (OAS1) mRNA expression. The most encouraging data was the high increment of serum neopterin, 8.0 ng/mL at 48 h, not been described before for any unmodified or pegylated IFN. Additionally, ß2M concentration doubled the pre-dose value at 24-48 hours. For both variables the values remained clearly upper baseline levels at 96 hours. CONCLUSIONS: HeberPAG possesses improved pharmacodynamic properties that may be very useful in the oncologic setting. Efficacy trials can be carried out to confirm these findings. TRIAL REGISTRATION: Registro Público Cubano de Ensayos Clínicos RPCEC00000130.

A vaccination strategy consisting of Semliki-Forest-virus (SFV) DNA prime and fowlpox-virus boost significantly protects mice from a recombinant (HIV-1) vaccinia-virus infection.

The use of DNA vectors based on the SFV (Semliki Forest virus) replicon have not been reported in the modality of DNA prime virus boost. In the present study, SFV DNA vectors (DNA vectors based on the SFV replicon) bearing the HIV-1 TAB9 multiepitopic polypeptide minigene were evaluated as priming DNA immunogens followed by a recombinant fowlpox expressing the TAB9 mutiepitope (FPTAB9LZ) boost. The results indicated that mice primed with pSFV(k)tab9 and boosted with FPTAB9LZ significantly decreased the HIV-1 recombinant (VVTAB13, a recombinant vaccinia virus expressing the TAB13 multiepitope) vaccinia virus replication, compared with groups given pSFV(k)tab9 vector and FPTAB9LZ virus alone. Additionally, the viral titre in ovary correlated with the number of specific gamma-interferon-secreting T-cells in spleen. These results support the possible use of SFV DNA vectors in prime-boost approaches implemented in therapeutic/prophylactic treatments for infectious diseases such as HIV-1.

Modified-vaccinia-virus-Ankara (MVA) priming and fowlpox-virus booster elicit a stronger CD8+ T-cell response in mice against an HIV-1 epitope than does a DNA/poxvirus prime-booster approach.

A prime-boost strategy combining FWPV (fowlpox virus) and the MVA (modified vaccinia virus Ankara), both expressing HIV-1 multi-V3 epitope polypeptides, was compared with a DNA-based Semliki Forest virus replicon/poxvirus approach for the induction of a CD8(+) T-cell response. Priming mice with recombinant MVA and boosting with recombinant FWPV, and not in the reverse order, increased the number of specific interferon-gamma-secreting cells in relation to the homologous combinations. Moreover, the improvement of the CD8(+) T-cell response with this combination was remarkably higher than that obtained by priming with a DNA vector containing a Semliki Forest virus replicon expressing the multi-epitope polypeptide and boosting either with recombinant MVA or FWPV. These results open a new and attractive alternative for vaccine preparation against HIV-1 using different immunogens.

The HIV-1 chimeric protein CR3 expressed by poxviral vectors induces a diverse CD8+ T cell response in mice and is antigenic for PBMCs from HIV+ patients.

Recombinant avipoxvirus vectors are attractive for vaccination against human immunodeficiency virus type 1 (HIV-1), where induction of a cytotoxic CD8(+) T cell (CTL) response seems to be an important component of protective immunity. We expressed the chimeric protein CR3, composed by CTL epitopes rich regions from, RT, Gag and Nef and conserved Th cell epitopes from gp120, gp41 and Vpr of HIV-1 in a fowlpox virus (FWPV) vector (FPCR3), and used this vector to induce HIV-specific CTL responses in mice. Mice immunised twice intraperitoneally with FPCR3, developed a CD8(+) T cell response measured as production of IFN-gamma by splenocytes in response to stimulation with P815 cells infected with recombinant vaccinia viruses (rVV) expressing CR3, Gag and Nef. The number of IFN-gamma secreting cells was markedly higher when a P815 cell line constitutively expressing CR3 was used as target cells for Enzyme-linked-immunospot (ELISPOT). CR3 epitopes were also specifically recognised by human PBMCs from three HIV(+) patients with different haplotypes. These results confirm the potential of FWPV vectors expressing these novel HIV-1 chimeric proteins to induce a simultaneous CD8(+) T cell response against conserved viral targets and early expressed regulatory proteins.

Effect of promoters on cellular immune response induced by recombinant fowlpox virus expressing multi-epitope polypeptides from HIV-1.

HIV stimulates strong immune CD8(+) cytotoxic T lymphocytes (CTL) response in infected people, despite causing an immunodeficiency. It has been demonstrated that this response could be very important for the control of the virus. We have shown previously that a recombinant fowlpox virus (rFWPV), expressing the multi-epitope polypeptide (MEP) from HIV-1 TAB9, induces strong and protective Th1 and CTL responses in Balb/c mice. Here, we have studied the CTL response against MEPs TAB9 and CR3 after immunizing with rFWPVs, where these genes are under the control of a strong synthetic early/late promoter or the 7.5 kDa promoter from vaccinia virus. TAB9 expression was increased by more than 9-fold using the strong promoter, which was translated into a two times increase in CTL response. The overall expression of CR3 was already ten times higher when compared with TAB9 with the 7.5 kDa promoter, but the use of a stronger promoter showed no effect either on the expression or CTL response. Moreover, rFWPV expressing TAB9 induced a stronger CTL response than those expressing CR3, measured as the number of interferon- gamma -secreting splenocytes, in spite of its lower antigen expression levels. These results suggest that the capacity of a stronger promoter to increase the MEP expression and/or CTL response against their epitopes is highly dependent on the nature of the polypeptide used.

Induction of a strong HIV-specific CD8+ T cell response in mice using a fowlpox virus vector expressing an HIV-1 multi-CTL-epitope polypeptide.

Recombinant avipoxvirus vectors are attractive candidates for use in vaccination strategies for infections such as human immunodeficiency virus type 1 (HIV-1), where induction of a CD8+ T cell response is thought to be an important component of protective immunity. Here, we report the expression of a multiepitope polypeptide (TAB9) composed of the central 15 amino acids of the V3 loop from six different isolates of HIV-1 in a fowlpox virus (FWPV) vector, and the use of this vector (FPTAB9LZ) to induce strong HIV-specific CD8+ T cell responses in mice. In animals immunized twice intravenously with FPTAB9LZ, almost 2% of the CD8+ T cells in the spleen were shown to produce IFN-gamma in response to stimulation with HIV-1 peptides 1 week after the second immunization. The most dominant response was to the HIV-1 IIIB peptide. A strong HIV-specific response was also induced by intraperitoneal immunization of mice with FPTAB9LZ, whilst subcutaneous immunization elicited a weaker response. Intraperitoneal immunization with FPTAB9LZ was also shown to provide protection against challenge with a recombinant vaccinia virus expressing antigens, including those in TAB9. These results confirm the potential of FWPV vectors for use in HIV vaccination strategies.