Sonia González

Safety and preliminary immunogenicity of the recombinant outer membrane protein P64k of Neisseria meningitidis in human volunteers

P64k is a meningococcal protein from Neisseria meningitidis that has been obtained by recombinant DNA technology. Recombinant P64k has been extensively characterized by physicochemical and immunological methods. Lately this protein has been found to act as a versatile immunological carrier for weak antigens in mice. In the present work, a Phase I clinical trial was carried out in healthy volunteers who received three inoculations of either placebo or recombinant P64k (20 or 50 μg). No severe adverse events occurred during the trial. Only mild adverse events in ten volunteers were observed. At 1 month after the third dose, 15 out of 18 volunteers (83.3%) who received the recombinant antigen had a P64k‐specific antibody titre 1:100, as detected by ELISA. A fourth dose, given 9 months after the third one, elicited a potent booster immune response in P64k vaccinees. Accordingly, these P64k formulations were considered safe and immunogenic in healthy human volunteers.

Effect of conjugation methodology on the immunogenicity and protective efficacy of meningococcal group C polysaccharide–P64k protein conjugates

Neisseria meningitidis serogroup C polysaccharide (CCPS) was conjugated to the carrier protein P64k using two different conjugation procedures, condensation mediated by carbodiimide with adipic acid dihydrazide as spacer and the reductive amination method. BALB/c mice were immunized with the resultant polysaccharide-protein conjugates and the immune response was evaluated. All conjugates assayed generated at least 10-fold higher antibody titers than the free polysaccharide. The reductive amination method rendered the best conjugate (CCPS-P64kR) that was able to elicit antibody titers statistically higher than the titer elicited by the plain CCPS (P<0.001). The sera of the group immunized with CCPS-P64kR showed a three-fold higher bactericidal response than the sera of the group immunized with the plain CCPS and they were able to protect against challenge with meningococci in the infant rat protection model. In addition, three different conjugates were obtained from polysaccharides with molecular relative sizes of 2000-4000 Da, 4000-10,000 Da or 10,000-50,000 Da, but no differences were detected in the immune response obtained against the three conjugates. Our experiments demonstrate that it is possible to generate a protective, T-cell-dependent response against CCPS using the P64k protein as carrier.

Expression in Escherichia coli of the lpdA gene, protein sequence analysis and immunological characterization of the P64k protein from Neisseria meningitidis

By making use of recombinant DNA technology it is possible to characterize meningococcal outer membrane proteins (OMPs) capable of stimulating a host immune response. The lpdA gene, which codes for an OMP (P64k) from Neisseria meningitidis, was cloned in Escherichia coli. The recombinant protein was recognized by sera from patients convalescing from meningococcal disease. The monoclonal antibodies obtained against the recombinant protein recognized the natural protein on a Western blot, and monoclonal antibody 114 was assayed in ELISA with a panel of 85 N. meningitidis strains. The protein was recognized in 81 strains (95.3%); the strains that were not recognized were neither epidemic nor isolated from systemic disease. The complete amino acid sequence of P64k was obtained by automatic sequencing and MS.

Genetic influence of the nonclassical major histocompatibility complex class I molecule MICB in multiple sclerosis susceptibility.

It has been widely reported that the major histocompatibility complex (MHC) class II region provides the main genetic contribution to multiple sclerosis (MS) susceptibility. However, recent studies have suggested that the MHC class I region may also contribute to the development of MS. In this study, we investigated the possible association of the human leukocyte antigen (HLA)-B, MHC class I chain-related gene B (MICB) and MHC class I chain-related gene A (MICA) genes, located in the MHC class I region, with MS susceptibility. For this purpose, we analyzed the distribution of HLA-DR, HLA-B, MICB and MICA alleles in 121 MS patients and 156 healthy controls. Neither HLA-B nor MICA alleles were found to be associated with MS susceptibility, and only the frequency of HLA-DRB1*01 allele was found to be increased in controls (31% vs 14%, P(c) = 0.011). However, MICB*004 allele frequency was significantly increased in MS patients (46.3% vs 23.3%, P(c) < 0.001, odds ratio = 2.82, 95% confidence interval = 1.68-4.73). Although, MICB*004 and HLA-DRB1*15 belong to the AH 7.1 ancestral haplotype, the association of MICB*004 to MS susceptibility was found to be independent of HLA-DRB1*15 in our population. This and previous studies clearly suggest that the MHC class I, in addition to class II, could be involved in MS susceptibility.

Recombinant Opc meningococcal protein, folded in vitro, elicits bactericidal antibodies after immunization

The meningococcal Opc protein has been expressed as inclusion bodies in Escherichia coli. After cell disruption and successive washing of the insoluble fraction, insoluble proteins were solubilized in presence of the chaotropic agent guanidium hydrochloride. The extract was applied to a Reverse Phase High Performance Liquid Chromatography (RP-HPLC)-C4 column, for further purification. The obtained recombinant Opc protein was refolded in vitro, by the addition of several compounds to the resuspended solution. Over time, the progress of renaturation was tested by immunoblot with the human monoclonal antibody LuNm03 against the meningococcal Opc protein. LuNm03 recognizes a conformational epitope on the native meningococcal Opc protein. Having established the optimal conditions of renaturation. Balb/c mice were immunized to study the humoral immune response. The human at immune response elicited in mice was measured by ELISA and immunoblot, while the functional activity of these antibodies was assayed in a bactericidal test. According to our results, it was possible to obtain a recombinant Opc protein folded in vitro, with a conformation suitable enough to generate functional antibodies in mice, capable of killing meningococci in the presence of human complement.

Assessment of vaccine potential of the Neisseria-specific protein NMB0938

The availability of complete genome sequence of Neisseria meningitidis serogroup B strain MC58 and reverse vaccinology has allowed the discovery of several novel antigens. Here, we have explored the potential of N. meningitidis lipoprotein NMB0938 as a vaccine candidate, based on investigation of gene sequence conservation and the antibody response elicited after immunization in mice. This antigen was previously identified by a genome-based approach as an outer membrane lipoprotein unique to the Neisseria genus. The nmb0938 gene was present in all 37 Neisseria isolates analyzed in this study. Based on amino acid sequence identity, 16 unique sequences were identified which clustered into three variants with identities ranging from 92 to 99%, with one cluster represented by the Neisseria lactamica strains. Recombinant protein NMB0938 (rNMB0938) was expressed in Escherichia coli and purified after solubilization of the insoluble fraction. Antisera produced in mice against purified rNMB0938 reacted with a range of meningococcal strains in whole-cell ELISA and western blotting. Using flow cytometry, it was also shown that anti-rNMB0938 antibodies bound to the surface of the homologous meningococcal strain and activated complement deposition. Moreover, antibodies against rNMB0938 elicited complement-mediated killing of meningococcal strains from both sequence variants and conferred passive protection against meningococcal bacteremia in infant rats. According to our results, NMB0938 represents a promising candidate to be included in a vaccine to prevent meningococcal disease.

B‐cell epitope mapping of the Neisseria meningitidis P64k protein using overlapping peptides

A common meningococcal antigen designated P64k has been identified, cloned and expressed in Escherichia coli. The recombinant antigen is highly immunogenic in several animal species and its immunogenicity in healthy human volunteers is under investigation. Recently, P64k has been used as an immunological carrier for weak immunogens. To characterize the B‐cell epitopes on P64k, recognized by immune sera obtained from mice, rabbits and monkeys, multiple overlapping peptides were synthesized and screened for antibody binding. Peptides covering the complete sequence of the P64k protein, 59 in all, of 20 amino acids each (overlapped by 10 residues), were synthesized. A number of continuous epitopes were detected with all sera, when immune and pre‐immune bleeds were compared. For mouse and monkey sera, a few major antigenic peptides were identified, while the recognition of the rabbit serum was much more heterogeneous. Despite variation in the exact location of continuous epitopes defined by different anti‐P64k sera, we found an immunogenic core region within the molecule, composed of amino acids Asp524–Gly533. Consistently, in this protein segment there was an amino acid stretch located in a β‐hairpin loop, which is exposed to the solvent in the previously determined three‐dimensional structure of the protein. This region is protruding and accessible to a sphere with a radius of 9 Å.

Preventive treatments for breast cancer: recent developments

Abstract Breast cancer is a burden for western societies, and an increasing one in emerging economies, because of its high incidence and enormous psychological, social, sanitary and economic costs. However, breast cancer is a preventable disease in a significant proportion. Recent developments in the armamentarium of effective drugs for breast cancer prevention (namely exemestane and anastrozole), the new recommendation from the National Institute for Health and Care Excellence to use preventative drugs in women at high risk as well as updated Guidelines from the US Preventive Services Task Force and the American Society of Clinical Oncology should give renewed momentum to the pharmacological prevention of breast cancer. In this article we review recent major developments in the field and examine their ongoing repercussion for breast cancer prevention. As a practical example, the potential impact of preventive measures in Spain is evaluated and a course of practical actions is delineated.

A deeper mining on the protein composition of VA-MENGOC-BC®: An OMV-based vaccine against N. meningitidis serogroup B and C

ABSTRACT The protein composition of an Outer Membrane Vesicle (OMV) preparation that constitutes the active pharmaceutical ingredient of VA-MENGOC-BC®, an effective vaccine against Neisseria meningitidis serogroups B, and C is presented. This preparation has a high lipid content and five abundant membrane proteins (FetA, PorA, PorB, RmpM, and Opc), constituting approximately 70% of the total protein mass. The protein composition was determined by combining the use of the Hexapeptide Ligand Library and an orthogonal tandem fractionation of tryptic peptides by reverse-phase chromatography at alkaline and acid pH. This approach equalizes the concentration of tryptic peptides derived from low- and high-abundance proteins as well as considerably simplifying the number of peptides analyzed by LC-MS/MS, enhancing the possibility of identifying low-abundance species. Fifty-one percent of the proteins originally annotated as membrane proteins in the genome of the MC58 strain were identified. One hundred and sixty-eight low-abundance cytosolic proteins presumably occluded within OMV were also identified. Four (NadA, NUbp, GNA2091, and fHbp), out of the five antigens constituting the Bexsero® vaccine, were detected in this OMV preparation. In particular, fHbp is also the active principle of the Trumenba® vaccine developed by Pfizer. The HpuA and HpuB gene products (not annotated in the MC58 genome) were identified in the CU385 strain, a clinical isolate that is used to produce this OMV. Considering the proteins identified here and previous work done by our group, the protein catalogue of this OMV preparation was extended to 266 different protein species.