Center for Genetic Engineering and Biotechnology

The control of classical swine fever (CSF) in endemic areas has been attempted with modified live vaccines. However, in some regions, the implementation of imperfect vaccination programs has led to a reduction in the genetic diversity of the circulating CSF virus (CSFV) strains and a change in their virulence. Porvac® subunit vaccine has been shown to provide a rapid onset of protection against the “Margarita” strain. The aim of this study was to evaluate whether the immune response induced by Porvac® is also effective against autochthonous CSFV isolates of low, medium or high virulence. All pigs vaccinated with Porvac® were protected against the disease after challenge. PR-11/10–3 isolate caused a very mild disease in controls, whilst Holguin_2009 isolate produced mild to moderate signs of CSF and one of the pigs died. Finally, controls inoculated with PR-2016 isolate developed moderate to severe signs of CSF and two of them died. Viral replication was detected in controls, but not in pigs immunized with Porvac®. Finally, anti-Erns antibodies were induced in five out of six control pigs but not in any of the vaccinated pigs. These results support the use of Porvac® for the control and elimination of CSF in Cuba and other endemic regions.

Cockroaches serve as mechanical vectors for medically important pathogens, and their presence in hospitals is a common occurrence. This review summarizes the pathogens carried by cockroaches collected in hospitals around the world during the period 2000–2024 and focuses on their antibiotic resistance mechanisms and potential impact on the public health system. The conventional techniques are most used to identify microorganisms and determine antibiotic resistance, but there are few studies that use molecular techniques for bacterial identification and resistance mechanism detection. The species that appear most frequently in the selected articles were Escherichia coli (22 articles) and Pseudomonas aeruginosa (11 articles). Regarding antibiotic resistance, this review describes 79.0% (34/43) of the studies analyzed. E. coli and P. aeruginosa bacteria were found to be resistant to antibiotics in 51.2% and 25.6% of articles, respectively. The identification of pathogens carried by cockroaches collected in hospitals suggests a potential risk of these insects in the transmission of healthcare-associated infections, mainly in developing countries, where this issue is most alarming. The collected data suggest that integrated approaches to cockroach control and infestation management should be put in place based on scientific evidence.

Classical swine fever (CSF) is endemic in Cuba and is one of the major health problems of the Cuban swine industry. The current efforts to control the disease in Cuba include vaccination with Porvac®, a subunit marker vaccine. Although the efficacy of Porvac against CSF virus (CSFV) subgenotype 1.4 has been extensively documented, little is known about the ability of the antibodies induced by this vaccine to neutralize other CSFV genotypes. In this study, sera collected from three pigs vaccinated with Porvac were able to efficiently neutralize CSFV strains belonging to genotypes 1, 2, and 3. The findings from this study indicate that additional in vivo studies are warranted to confirm the ability of this vaccine to protect pigs against CSFV genotypes 2 and 3.

Background Classical Swine Fever (CSF) is still one of the most economically important viral diseases of pigs. In endemic countries, the disease is controlled mostly through vaccination; hence, the availability of safe and effective vaccines is of utmost importance. Vaccines intended for application in developing countries must also be thermally stable, since the infrastructure needed to maintain a cold chain in those countries is usually lacking. Porvac® is a second-generation subunit marker vaccine against CSF that has demonstrates to be safe and protective. Previous studies have also shown that the vaccine is stable for 1 week at 37 oC and have a shelf life of at least 36 months at 2–8 oC. The aim of this work was to further explore the accelerated stability of Porvac® by assessing the physicochemical properties of the emulsion, and the safety and immunogenicity of the vaccine subjected to more drastic conditions of thermal stress: (1) 25 oC for 12 months; (2) 30oC and 37 oC for one month and (3) 15 days at 37 °C after the cap of the vials had been needle-punctured. Results The vaccine subjected to all these conditions did not show significant changes in the physicochemical properties of the emulsion; did not produce local or systemic adverse reactions in pigs, and the chromatographic profile of the recovered antigen was preserved. All vaccinated swine developed neutralizing antibody titers ≥ 1:1000 at 28 days post vaccination. Conclusions Porvac® is stable in all the experimental conditions tested, even after cap puncture, and retains the capacity to induce high titers of neutralizing antibodies, well above the threshold of protection. These results reinforce the robustness of the vaccine, and support its use as a very attractive alternative to modified live vaccines in developing countries endemic for CSF.

Purpose The next-generation anti-tumor drug peptide CIGB-300, developed by the Center for Genetic Engineering and Biotechnology (CIGB), targets casein kinase 2 (CK2) and its substrates, implicating significant therapeutic potential in cancer treatment. A key focus of this study was to compare CIGB-300 and a primary synthetic byproduct, CIGB-300iso, which shares the amino acid sequence with CIGB-300 but was proposed to differ due to racemization. Methods This study explores the synthesis, characterization, and structural elucidation of CIGB-300 and its isomer CIGB-300iso by a comprehensive NMR analysis of seven synthesized diastereomers including amino acid residues C15, H21, and C25. Results This study revealed that CIGB-300iso contains one D enantiomer at position H21. The structures of both isoforms derived from NMR constraints disclosed that the L and D enantiomers have an altered peptide supersecondary structure, with a β-turn type IV3 found in CIGB-300 and a type I β-turn in CIGB-300iso. Conclusion The configuration of H21 significantly impacts the peptide’s conformations, sidechain orientations and, potentially, its biological activity. These findings highlight the importance of enantiomerically pure peptides for the design and synthesis of drug peptides.

Properties of recombinant glycoproteins can be altered by the addition of oligosaccharide structures specific to the cells used for its heterologous expression. A methodology was implemented to obtain a glycopeptide preparation useful to elucidate the role of carbohydrates in the immunogenicity and antigenicity of glycoproteins. It consists on the digestion of the protein, followed by selective capture of the oligosaccharides bound to di-/tripeptides, and their grafting onto a non-glycosylated receptor protein by chemical crosslinking. Glycopeptides derived from C-RBD-H6 PP protein, the active ingredient of the Abdala vaccine were efficiently grafted onto a non-glycosylated protein as evidenced by western blotting. 10/30/2024-Open Access

Keyhole limpet haemocyanins (KLH1 and KLH2) from Megathura crenulata, are large (approx. 3900 amino acids) multi-subunit oxygen-carrying metalloproteins, that are widely used as carrier proteins in conjugate vaccines and in immunotherapy. KLHs and their derived conjugate vaccines are poorly characterized by mass spectrometry due to the very stable supramolecular structures with megadalton molecular mass, and the resistance to be efficiently digested with standard protocols using specific proteases. KLH1 and KLH2 proteins were conjugated to the conserved twenty-one amino acids pP0 peptide, derived from the P0 acidic ribosomal protein of Rhipicephalus sp. ticks by using the maleimide-thiol chemistry. The resultant KLH1- and KLH2-Cys1pP0 conjugate vaccines were efficiently digested using the MED-FASP procedure, and analyzed by LC-MS/MS allowing approximately 85% sequence coverage of both conjugates. New PTMs not described for the KLH such as oxidized species were identified. The conjugation sites and Cys-His thioether bonds were determined by identifying cross-linked peptides using software developed for cross-linking mass spectrometry experiments. Conjugation sites were validated by combining sequence coverage, the presence of diagnostic and linker fragment ions in the MS/MS spectra, and the multi-peak pattern in the extracted ion chromatograms. In summary, 124 out of 170 Lys (75%) and 99 out 150 Lys (66%) were found conjugated to Cys1pP0 in KLH1 and KLH2, respectively. In total, four and seven Cys-His thioether bonds were experimentally determined in KLH1 and KLH2, respectively. This is the first report of the conjugation sites identification of two KLH-based vaccines determined by mass spectrometry.

The peptide CIGB‐210 inhibits HIV replication, inducing a rearrangement of vimentin intermediate filaments. The assessment of the in vitro serum and plasma stability of this peptide is important to develop an optimal pharmacological formulation. A half‐life of 17.68 ± 0.59 min was calculated for CIGB‐210 in human serum by reverse‐phase high‐performance liquid chromatography (HPLC) and mass spectrometry (MS). Eight metabolites of CIGB‐210 were identified with this methodology, all of them lacking the N‐terminal moiety. A previously developed CIGB‐210 in‐house competitive ELISA was used to compare the stability of CIGB‐210 derivatives containing either D‐amino acids, acetylation at the N‐terminus, or both modifications. The half‐life of CIGB‐210 in serum was five times higher when measured by ELISA than by HPLC/MS, and twice higher in plasma as compared to serum. The substitution of D‐asparagine on position 6 doubled the half‐life, while D‐amino acids on positions 8 and 9 did not improve the stability. The acetylation of the N‐terminus resulted in a 24‐fold more stable peptide in plasma. The positive effect of N‐terminal acetylation on CIGB‐210 serum stability was confirmed by the HPLC/MS method, as the half‐life of the peptide was not reached after 2 h of incubation, which represents more than a 6.8‐fold increase in the half‐life with respect to the original peptide.

A phase 1–2, prospective, multicenter, randomized, open-label clinical trial (Code RPCEC00000382), with parallel groups, involving 1161 participants, was designed to assess the safety and immunogenicity of two Cuban COVID-19 vaccines (Mambisa and Abdala) in boosting COVID-19 immunity of convalescent adults after receiving one dose of either vaccine. The main safety outcome was severe vaccination adverse events occurring in <5% of vaccinees. Main immunogenicity success endpoints were a ≥4-fold anti-RBD IgG seroconversion or a ≥20% increase in ACE2-RBD inhibitory antibodies in >55% of vaccinees in Phase 1 and >70% in Phase 2. Neutralizing antibody titers against SARS-CoV-2 variants were evaluated. Both vaccines were safe—no deaths or severe adverse events occurred. Mild intensity adverse events were the most frequent (>73%); headaches predominated for both vaccines. Phase 1 responders were 83.3% (p = 0.0018) for Abdala. Mambisa showed similar results. Phase 2 responders were 88.6% for Abdala (p < 0.0001) and 74.2% for Mambisa (p = 0.0412). In both phases, anti-RBD IgG titers, inhibition percentages and neutralizing antibody titers increased significantly after the booster dose. Both vaccines were safe and their immunogenicity surpassed the study endpoints.

The Hepatitis B core antigen (HBcAg) has been used as a carrierof several heterologous protein fragments based on its capacity to form virus-like particles (VLPs)and to activate innate and adaptive immune responses. In the present work, two chimeric proteins were designed as potential pancorona vaccine candidates, comprising the N- or C- terminal domain of SARS-CoV-2 nucleocapsid (N) protein fused to HBcAg. The recombinant proteins, obtained in E. coli , were named CN-1 and CND-1, respectively. The final protein preparations were able to form 10-25 nm particles, visualized by TEM. Both proteins were recognized by sera from COVID-19 convalescent donors; however,the antigenicity of CND-1 tends to be higher. The immunogenicity of both proteins was studied in Balb/C mice by intranasal route without adjuvant. After three doses, only CND-1 elicited a positive immune response, systemic and mucosal, against SARS-CoV-2 N protein. CND-1 was evaluated in a second experiment mixed with the CpG ODN-39M as nasal adjuvant. The induced anti-N immunity was significantly enhanced, and the antibodies generated were cross-reactive with N protein from Omicron variant, and SARS-CoV-1. Also, an anti-N broadcellular immune response was detected in spleen, by IFN-g ELISpot. The nasal formulation composed by CND-1 and ODN-39M constitutes an attractive component for a pancorona vaccine, by inducing mucosal immunity and systemic broad humoral and cellular responses against Sarbecovirus N protein.

The next-generation anti-tumor drug peptide CIGB-300, developed by the Center for Genetic Engineering and Biotechnology (CIGB), targets casein kinase 2 (CK2) and its substrates, implicating significant therapeutic potential in cancer treatment. A key focus of this study was to compare CIGB-300 and a primary synthetic byproduct, CIGB-300iso, which shares the amino acid sequence with CIGB-300 but was proposed to differ due to racemization. This study explores the synthesis, characterization, and structural elucidation of CIGB-300 and its isomer CIGB-300iso. A comprehensive NMR analysis of seven synthesized diastereomers including amino acid residues C15, H21, and C25 revealed that CIGB-300iso contains one D enantiomer at position H21. The structures of both isoforms derived from NMR constraints disclosed that the L and D enantiomers have an altered peptide supersecondary structure, with a β-turn type IV 3 found in CIGB-300 and a type I β-turn in CIGB-300iso, significantly impacting the peptide's conformations, sidechain orientations and, potentially, its biological activity. These findings highlight the importance of enantiomerically pure peptides for the design and synthesis of drug peptides.

Multiple sclerosis (MS) is an inflammatory, demyelinating, autoimmune disease of the central nervous system. It is known that the Major Histocompatibility Complex class II region produces the most potent effect on MS genetic susceptibility. In addition, the genetic polymorphism within the TNF locus has been involved in the pathogenesis of various autoimmune diseases. This study has the purpose of evaluating HLA-DRB1, HLADQB1 alleles and TNF promotor alpha gene polymorphism (SNP TNF- α -238 G/A; - 243G/A; -308 G/A; - 375 G/A, -856 C/T; -862 C/A) in a sample of Cuban MS patients. Disease-associated HLA susceptibility alleles were genotyped by the SSP-PCR method. The TNF- α genotypes were identified by sequencing. The association was found between HLA and MS, DRB1*15:01, DRB1*14:01, DQA*01:02 and DQB1*06:02 being susceptibility alleles. TNF-α-308 G (OR=1,6, P<0,01) and TNF- α -238 G (OR=2,0, P<0,01) alleles had higher frequency among MS patients than control subjects. The odds ratio was increased among HLADRB1*1501 positive individuals. Our results have shown that the combination of TNF-α-238 G, -308 G with HLA-DRB1*15:01 and HLA-DQA1*01:02 increased susceptibility to MS (p<0.05 OR=4.2) in the Cuban population. Keywords: HLA, TNF-Alpha, polymorphism, SNP, Multiple Sclerosis, Cuban population

Multiple sclerosis (MS) is an inflammatory, demyelinating, autoimmune disease of the central nervous system. It is known that the Major Histocompatibility Complex class II region produces the most potent effect on MS genetic susceptibility. In addition, the genetic polymorphism within the TNF locus has been involved in the pathogenesis of various autoimmune diseases. This study has the purpose of evaluating HLA-DRB1, HLA-DQB1 alleles and TNF promotor alpha gene polymorphism (SNP TNF- α -238 G/A; - 243G/A; -308 G/A; -375 G/A, -856 C/T; -862 C/A) in a sample of Cuban MS patients. Disease-associated HLA susceptibility alleles were genotyped by the SSP-PCR method. The TNF- α genotypes were identified by sequencing. The association was found between HLA and MS, DRB1*15:01, DRB1* 14:01, DQA*01:02 and DQB1*06:02 being susceptibility alleles. TNF-α-308 G (OR=1,6, P<0,01) and TNF- α -238 G (OR=2,0, P<0,01) alleles had higher frequency among MS patients than control subjects. The odds ratio was increased among HLADRB1*1501 positive individuals. Our results have shown that the combination of TNF-α-238 G, -308 G with HLA-DRB1*15:01 and HLA-DQA1*01:02 increased susceptibility to MS (p<0.05 OR=4.2) in the Cuban population. Keywords: HLA, TNF-Alpha, polymorphism, SNP, Multiple Sclerosis, Cuban population

The classical swine fever is endemic and a major health problem for the swine industry in Cuba. The current efforts to control the disease include vaccination with Porvac®, a subunit marker vaccine. Although the efficacy of Porvac ® against subgenotype 1.4 has been extensively documented, little is known about the abilty of the antibodies induced by this vaccine to neutralize other genotypes. Sera collected frm three pigs vaccinated with Porvac ® were able to efficiently neutralize CSFV strains belonging to genotypes 1, 2 and 3. Porvac ® -induced antibodies also neutralized bovine viral diarrhea virus and border disease virus. The results suggest that Porvac ® marker vaccine could be used for controlling CSF globally.

Introduction: Dilated cardiomyopathy (DCM) is a fatal myocardial condition with ventricular structural changes and functional deficits, leading to systolic dysfunction and heart failure (HF). DCM is a frequent complication in oncologic patients receiving Doxorubicin (Dox). Dox is a highly cardiotoxic drug, whereas its damaging spectrum affects most of the organs by multiple pathogenic cascades. Experimentally reproduced DCM/HF through Dox administrations has shed light on the pathogenic drivers of cardiotoxicity. Growth hormone (GH) releasing peptide 6 (GHRP-6) is a GH secretagogue with expanding and promising cardioprotective pharmacological properties. Here we examined whether GHRP-6 administration concomitant to Dox prevented the onset of DCM/HF and multiple organs damages in otherwise healthy rats. Methods: Myocardial changes were sequentially evaluated by transthoracic echocardiography. Autopsy was conducted at the end of the administration period when ventricular dilation was established. Semiquantitative histopathologic study included heart and other internal organs samples. Myocardial tissue fragments were also addressed for electron microscopy study, and characterization of the transcriptional expression ratio between Bcl-2 and Bax. Serum samples were destined for REDOX system balance assessment. Results and discussion: GHRP-6 administration in parallel to Dox prevented myocardial fibers consumption and ventricular dilation, accounting for an effective preservation of the LV systolic function. GHRP-6 also attenuated extracardiac toxicity preserving epithelial organs integrity, inhibiting interstitial fibrosis, and ultimately reducing morbidity and mortality. Mechanistically, GHRP-6 proved to sustain cellular antioxidant defense, upregulate prosurvival gene Bcl-2, and preserve cardiomyocyte mitochondrial integrity. These evidences contribute to pave potential avenues for the clinical use of GHRP-6 in Dox-treated subjects.