Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X

Neisseria meningitidis is a major cause of bacterial meningitis worldwide, especially in the African meningitis belt, and has a high associated mortality. The meningococcal serogroups A, W, and X have been responsible for epidemics and almost all cases of meningococcal meningitis in the meningitis belt over the past 12 y. Currently no vaccine is available against meningococcal X (MenX). Because the development of a new vaccine through to licensure takes many years, this leaves Africa vulnerable to new epidemics of MenX meningitis at a time when the epidemiology of meningococcal meningitis on the continent is changing rapidly, following the recent introduction of a glycoconjugate vaccine against serogroup A. Here, we report the development of candidate glycoconjugate vaccines against MenX and preclinical data from their use in animal studies. Following optimization of growth conditions of our seed MenX strain for polysaccharide (PS) production, a scalable purification process was developed yielding high amounts of pure MenX PS. Different glycoconjugates were synthesized by coupling MenX oligosaccharides of varying chain length to CRM₁₉₇ as carrier protein. Analytical methods were developed for in-process control and determination of purity and consistency of the vaccines. All conjugates induced high anti-MenX PS IgG titers in mice. Antibodies were strongly bactericidal against African MenX isolates. These findings support the further development of glycoconjugate vaccines against MenX and their assessment in clinical trials to produce a vaccine against the one cause of epidemic meningococcal meningitis that currently cannot be prevented by available vaccines.A major cause of bacterial meningitis worldwide, Neisseria meningitidis has significant associated mortality (1). Among the 13 distinct meningococcal serogroups, which are classified on the structure of their capsular polysaccharide (PS), serogroups A, B, C, Y, W, and X most commonly cause invasive disease, including meningitis and septicemia, in humans. The highest incidence of meningococcal meningitis occurs in the meningitis belt of sub-Saharan Africa, extending from Senegal to Ethiopia.Since records began, meningococcal serogroup A (MenA) has been the dominant cause of epidemics of meningococcal meningitis in this region (2), but MenW (3) and MenX (4–6) have also been responsible for epidemics. From 2010 to 2012, MenX was responsible for annual meningitis outbreaks in Burkina Faso. In 2011, MenX accounted for 59% of confirmed cases of meningococcal meningitis in this country (7). Higher case fatality rates have been reported for meningitis caused by MenX compared with MenA (4, 6), and children aged 1–9 y constitute the most affected age group (4, 8).In 2010, a MenA conjugate vaccine (MenAfriVac) was rolled out in a mass vaccination program in Burkina Faso, Mali, and Niger (9). Early reports indicate that this has been highly effective at reducing cases of MenA meningitis. Removal of serogroup A strains from circulating among the population may confer an advantage to MenX, previously less able to compete with the more virulent serogroup A (10, 11). Capsule replacement of carried meningococci did not occur following the implementation of serogroup C conjugate vaccines in the United Kingdom (12). However, the conditions in the meningitis belt are very different from those in industrialized nations and a recent study of carriage before and after the introduction of the MenA conjugate vaccine in Burkina Faso found significantly higher levels of MenX carriage following the introduction of the vaccine (13).MenW PS vaccine is used for outbreak control of meningitis caused by MenW in the meningitis belt and a change in the epidemiology of meningitis due to MenW could necessitate its increased demand. Polyvalent vaccines, including MenW glycoconjugate, are currently produced and used in developed countries and could potentially be mobilized for use in Africa. In contrast, although the need for a vaccine against serogroup X Neisseria meningitidis has been recognized for many years (4, 5, 14, 15), none is currently available.Given the success of other meningococcal glycoconjugate vaccines (16), the MenX PS antigen is a logical target for vaccine design. Plain PS could facilitate epidemic control, whereas conjugation to a carrier protein would provide enhanced immunogenicity, particularly from early infancy, by converting the PS into a T-cell–dependent antigen (17, 18). As recognized for other PS, conjugation to an appropriate carrier protein overcomes the limits of PS vaccines, such as poor efficacy in children less than 2 y, lack of immunological memory with poor booster responses, and relatively short duration of protection (19–21). Meningococcal conjugate vaccines are also able to overcome the immune hyporesponsiveness that is induced by PS vaccines (22, 23). Additionally, as documented for group C, meningococcal conjugate vaccines can reduce carriage of N. meningitidis in the nasopharynx, decreasing transmission (24), whereas PS vaccines have not been shown to provide substantial herd immunity (25). The impact of vaccination with the MenAfriVac conjugate vaccine in Burkina Faso on carriage and herd immunity has been recently reported (13).The MenX PS was first characterized and defined as a distinct serogroup in the 1960s (26, 27) and was shown to be immunogenic in rabbits (28, 29). The structure of MenX PS consists of N-acetylglucosamine-4-phosphate residues held together by α-(1-4) phosphodiester bonds without O-acetyl groups (28, 30–33).Here, we describe the process of synthesis of MenX glycoconjugate vaccines, together with data from preliminary immunological evaluation in mice. MenX oligosaccharides (OS) of different length and three different conjugation chemistries were compared, using CRM_197, a nontoxic mutant of diphtheria toxin (34), as carrier protein. When tested in mice, all MenX–CRM₁₉₇ conjugates resulted in high IgG antibody levels and serum bactericidal activity (SBA) titers, indicating the path ahead for the clinical development of a vaccine to prevent the remaining cause of epidemic meningococcal meningitis in Africa for which no vaccine is available.     

Genetic Variability in Platelet Integrin α2β1 Density: Possible Contributor to Plasmodium vivax–induced Severe Thrombocytopenia

Understanding the pathogenesis of Plasmodium vivax malaria is challenging. We hypothesized that susceptibility to P. vivax-induced thrombocytopenia could be associated with polymorphisms on relevant platelet membrane integrins: integrin α2 (C807T), and integrin β3 (T1565C). Although β3 polymorphism was not related with P. vivax malaria, α2 807T carriers, which show high levels of integrin α2β1, had a higher probability for severe thrombocytopenia than wild-type carriers. This evidence of the association of integrin polymorphism and P. vivax morbidity was further demonstrated by a moderate but significant correlation between clinical disease and surface levels of the integrin α2β1.Plasmodium vivax infection is no longer considered a benign disease because it might cause severe or fatal episodes.^1,2 Although the mechanisms underlying P. vivax-induced pathogenesis remain poorly studied, thrombocytopenia is frequently observed in P. vivax infection.³ Recent studies suggest an association between deep thrombocytopenia and severity of the illness.⁴ However, the mechanisms leading to thrombocytopenia, as well as its contribution to malaria pathogenesis, are not well understood.Besides their central role in homeostasis, platelets contain a wide range of inflammatory, immune-modulating, and angiogenic factors. Consequently, it is not surprising that the role of platelets in the development of an array of disorders continues to emerge.⁵ Although P. vivax-induced thrombocytopenia has not been investigated in detail,³ several lines of evidence suggest that platelets participate actively in the pathogenesis of malaria.⁶ In P. vivax malaria, platelets release microparticles into the circulation, and these platelet-derived microparticles seem to be associated with acute inflammatory symptoms of disease.⁷ In addition, we have shown that levels of plasma cell-free circulating nucleic acids were closely correlated with platelet counts, increasing in a linear fashion with the spectrum of P. vivax malaria.⁸ On the basis of these observations, we hypothesized that platelet receptor polymorphisms that result in a gain of function in platelet adhesion and/or aggregation in vivo might place carriers at increased risk for P. vivax-induced thrombocytopenia.We studied the association between P. vivax and polymorphisms of platelet integrins (cell-surface heterodimeric proteins that mediate cell-matrix and cell-cell interactions⁹). The focus of this study was two gain-of-function platelet receptor single-nucleotide polymorphisms: the C807T polymorphism of integrin α2 (also known as platelet glycoprotein Ia, GPIa) and the T1565C of integrin β3 (platelet glycoprotein IIIa, GPIIIa). Both polymorphisms have been implicated in different clinical events, including those related to the coronary syndromes, probable because of their gain-of-function mechanisms.^10,11 Integrin α2, a platelet receptor for collagen, forms a functional receptor with the integrin β₁ subunit, which is essential for platelet function.⁹ The C807T single nucleotide polymorphism (single-nucleotide polymorphism no. rs1126643; National Center for Biotechnology Information, Bethesda, MD) is considered a genetic marker of the integrin α2β1 density.^12,13 Integrin β3, a common β subunit for β3-integrins, such as αIIbβ3, has a key role in platelet function by binding fibrinogen and von Willebrand factor (vWF),¹⁴ and the T1565C polymorphism (rs5918) seems to increase platelet aggregation.¹⁵ To the best of our knowledge, there has been no study that assessed the association between integrin polymorphisms and P. vivax malaria.A total of 150 P. vivax patients 2–78 years of age were enrolled in the study after written informed consent, as specified by the Brazilian National Council of Health (Protocol CEPSH/CPqRR/03/2008). Antimalarial and supportive therapies were given according to standard protocols. The study included patients with symptomatic but uncomplicated P. vivax malaria, and all volunteers were negative for P. falciparum and/or P. malariae by microscopy and polymerase chain reaction. Demographic, clinico-epidemiologic, and hematologic data of P. vivax-infected volunteers are shown in

Endocannabinoids in chronic migraine: CSF findings suggest a system failure.

Based on experimental evidence of the antinociceptive action of endocannabinoids and their role in the modulation of trigeminovascular system activation, we hypothesized that the endocannabinoid system may be dysfunctional in chronic migraine (CM). We examined whether the concentrations of N-arachidonoylethanolamide (anandamide, AEA), palmitoylethanolamide (PEA), and 2-arachidonoylglycerol (2-AG) in the CSF of patients with CM and with probable CM and probable analgesic-overuse headache (PCM+PAOH) are altered compared with control subjects. The above endocannabinoids were measured by high-performance liquid chromatography (HPLC), and quantified by isotope dilution gas-chromatography/mass-spectrometry. Calcitonin gene-related peptide (CGRP) levels were also determined by RIA method and the end products of nitric oxide (NO), the nitrites, by HPLC. CSF concentrations of AEA were significantly lower and those of PEA slightly but significantly higher both in patients with CM and PCM+PAOH than in nonmigraineur controls (p<0.01 and p<0.02, respectively). A negative correlation was found between AEA and CGRP levels in CM and PCM+PAOH patients (r=0.59, p<0.01 and r=-0.65, p<0.007; respectively). A similar trend was observed between this endocannabinoid and nitrite levels. Reduced levels of AEA in the CSF of CM and PCM+PAOH patients may reflect an impairment of the endocannabinoid system in these patients, which may contribute to chronic head pain and seem to be related to increased CGRP and NO production. These findings support the potential role of the cannabinoid (CB)1 receptor as a possible therapeutic target in CM.     

Leuprorelin acetate affects ERK1/2 activity in prostate cancer cells

The mechanisms by which a GnRH analogue, leuprorelin acetate (LA), antagonizes the mitogenic effect of dihydrotestosterone (DHT) or epidermal growth factor (EGF) in prostate cancer cells is poorly understood. The mitogen-activated protein kinase system has a central role in growth regulation and, for this reason, we investigated the involvement of the extracellular signal-regulated kinase (ERK1/2) pathway in the response of both androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer cells to LA alone or combined with EGF or DHT. The evaluation of ERK activation was performed by using Western blot analysis and immunocytochemistry. EGF specifically induced ERK1/2 activity in both models and this effect was counteracted by an inhibitor of EGF-receptor phosphorylation. The addition of LA produced an appreciable reduction of ERK phosphorylation promoted by EGF in LNCaP cells, while it generally determined an increase in ERK activity in androgen-unresponsive PC-3 cells. The slight ERK activation induced by DHT in LNCaP cells was counteracted by LA and this effect was evident only by immunocytochemistry. Our findings suggest that the antiproliferative effect of LA in prostate cancer cells stimulated by hormones and growth factors may be, at least in part, mediated by the reduction of ERK1/2 activation in LNCaP cells and linked to the unexpected increase of this activity produced by the analogue in PC-3 cells.     

Role of the molecular staging and response in the management of follicular lymphoma patients

Bcl-2/IgH rearrangement is the molecular hallmark of follicular lymphoma which is present in 70 - 90% of cases at diagnosis. The significance of the bcl-2 rearrangement at onset of disease and of its clearing after treatment (molecular response) is still controversial.

The aims of the present analysis are: to evaluate the incidence of bcl-2 rearrangement in blood and marrow in a cohort of patients systematically investigated at diagnosis, to describe the correlation between bcl-2 and presenting features, to clarify the correlation of molecular response with outcome.

Of 98 patients studied at initial staging for the presence of bcl-2 rearrangement, 64 (65%) showed bcl-2/IgH rearrangement in peripheral blood (PB) and/or bone marrow (BM) (58 at Major Breakpoint Region, MBR, and 6 at minor cluster region, mcr) while no bcl-2/IgH rearrangement was detected in the remaining 34 (35%) (germline status). No statistically significant differences were found between bcl-2 positive and bcl-2 negative cases as concerns presenting clinical features and response to first-line therapy. The median event-free survival, EFS, was not reached for the bcl-2 negative patients in PB and was 11 months for bcl-2 positive patients (statistically significant, P = 0.01) and, similarly, the median EFS was not reached for the bcl-2 negative patients in BM and was 11 months for bcl-2 positive patients (statistically significant, P = 0.04).

Of the 64 bcl-2 positive cases, patients were analysed for molecular response (48 in BM and 40 in PB): 16 were molecular responders in BM and 20 were molecular responders in PB. The median EFS was 19 months for molecular responders in PB and 9 months for non-responders; 1-year-EFS was 68% (95% CI; 49 - 88), for responders in PB and 42% (95% CI; 22 - 61) for non-responders (P = 0.05). The median EFS was 11 months both for molecular responders and non-responders in BM; 1-year-EFS was 52% for responders in BM (CI; 30 - 73), and 43% (CI 33 - 71) for non-responders (P = 0.7). No clinical feature showed significant correlation with PB and BM molecular responses.

This analysis shows that bcl-2 rearrangement in blood and bone marrow is frequently detected at staging, even in stage I disease. Absence of the bcl-2 rearrangement is related to a better EFS and the achievement of a molecular response in peripheral blood after therapy is associated with a better EFS.     

Low titer maternal antibodies can both enhance and suppress B cell responses to a combined live attenuated human rotavirus and VLP-ISCOM vaccine

We investigated effects of low titer (Lo) circulating MatAb on protection and immunogenicity of attenuated (Att) human rotavirus (HRV) priming and 2/6-virus-like particle (VLP)-immunostimulating complex (ISCOM) boosting (AttHRV/VLP) or VLP-ISCOM alone vaccines. LoMatAb had both enhancing and suppressing effects on B cell responses, depending on tissue, antibody isotype and vaccine. Differential effects of LoMatAb on IgA responses in different tissues suggest that LoMatAb did not suppress induction of IgA effector and memory B cells but impaired homing of these cells to secondary lymphoid or effector tissues, reducing IgA antibody secreting cells and antibodies at these sites. The AttHRV/VLP vaccine partially overcame LoMatAb suppression, conferred moderate protection against virulent HRV (as measured by reduced viral shedding and diarrhea) and represents a new candidate for rotavirus vaccines for both humans and animals.     

Palpebral cryptococcosis: case report

This paper is about a patient with acquired immunodeficiency syndrome empirically treated for miliary tuberculosis. During the clinical evolution the patient presented lesions compromising the right eyelid and tarsal conjunctiva. The initial diagnostic hypothesis was ocular tuberculosis with conjunctival and eyelid involvement. The biopsy of the conjunctival lesion identified an encapsulated yeast-like fungus: Criptococcus neoformans. After starting treatment with B anfotericin, the cutaneus lesions cleared.     

Citicoline and lithium rescue retinal ganglion cells following partial optic nerve crush in the rat

Citicoline and lithium (Li(-)) have been shown to support retinal ganglion cell (RGC) survival and axon regeneration in vitro. Optic nerve crush (ONC) is a model of both brain axonal injury and certain aspects of the glaucomatous degeneration of RGC. We have used this model to quantify protection offered to RGC by these drugs and to determine whether their effects are mediated by enhanced expression of the antiapoptotic protein Bcl-2. Adult rats (6-12 per group) were subjected to ONC accompanied by a contralateral sham operation. Animals were treated intraperitoneally with either vehicle, citicoline sodium (1g/kg daily for up to 7 days and 300 mg/kg daily afterwards), lithium chloride (30 mg/kg daily), or both drugs combined. Fluorogold was injected bilaterally into superior colliculi 1, 5 or 19 days after ONC. Labeled cells were counted under a fluorescence microscope 2 days after tracer injection. In a separate set of experiments the effects of treatments on expression of Bcl-2 in retinas were evaluated by immunohistochemistry. In vehicle-treated animals there was a progressive decrease of RGC density after crush. This decrease was attenuated in citicoline-treated animals 1 week and 3 weeks after the crush. In the lithium-treated group protection was even more pronounced. In animals treated with both drugs RGC protection was similar to that achieved by lithium alone. Bcl-2 immunoreactivity was seen predominantly in retinal ganglion cells. Its increase was recorded in the lithium and citicoline group as well as in animals treated with the combination of both drugs. Both citicoline and lithium protect RGC and their axons in vivo against delayed degeneration triggered by the ONC. Retinoprotective action of both drugs may involve an increase in Bcl-2 expression.     

JAK2 (V617F) as an acquired somatic mutation and a secondary genetic event associated with disease progression in familial myeloproliferative disorders

BACKGROUND: A somatic gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been identified in chronic myeloproliferative disorders, which appear to have a sporadic occurrence in most individuals. The authors studied the biologic significance of the JAK2 (V617F) mutation in familial myeloproliferative disorders. METHODS: Twenty pedigrees with familial chronic myeloproliferative disorders were identified through an investigation of family history in 264 patients with sporadic myeloproliferative disorders. A quantitative real-time polymerase chain reaction (qRT-PCR)-based allelic discrimination assay was employed for the detection of the V617F mutation in circulating granulocytes and T lymphocytes. An analysis of X-chromosome inactivation pattern was performed in female patients. RESULTS: Fourteen families had homogeneous phenotypes, and 6 families had mixed phenotypes. By using a qRT-PCR-based allelic discrimination assay, the JAK2 (V617F) mutation was detected in circulating granulocytes from 20 of 31 patients, but the mutation was not detected in T lymphocytes. Granulocyte mutant alleles ranged from 2.1% to 91.5% and, on average, increased with time. Discordant distribution of the JAK2 (V617F) mutation was observed in siblings with polycythemia vera. The proportion of granulocytes that carried the JAK2 (V617F) mutation was lower than the proportion of clonal granulocytes, as determined in an analysis of X-chromosome inactivation patterns in female patients. CONCLUSIONS: The current findings indicated that the JAK2 (V617F) mutation represents an acquired somatic mutation in patients with familial chronic myeloproliferative disorders and probably occurs as a secondary genetic event in the background of preexisting clonal hematopoiesis. Thus, a genetic predisposition to acquisition of JAK2 (V617F) is inherited in families with myeloproliferative disorders.     

The anti-human leukocyte antigen-DR monoclonal antibody 1D09C3 activates the mitochondrial cell death pathway and exerts a potent antitumor activity in lymphoma-bearing nonobese diabetic/severe combined immunodeficient mice

The fully human anti-HLA-DR antibody 1D09C3 has been shown to delay lymphoma cell growth in severe combined immunodeficient (SCID) mice. The present study was aimed at (a) investigating the mechanism(s) of 1D09C3-induced cell death and (b) further exploring the therapeutic efficacy of 1D09C3 in nonobese diabetic (NOD)/SCID mice. The chronic lymphocytic leukemia cell line JVM-2 and the mantle cell lymphoma cell line GRANTA-519 were used. Generation of reactive oxygen species (ROS) and mitochondrial membrane depolarization were measured by flow cytometry following cell incubation with dihydroethidium and TMRE, respectively. Western blot analysis was used to detect c-Jun-NH(2)-kinase (JNK) phosphorylation and apoptosis-inducing factor (AIF). NOD/SCID mice were used to investigate the activity of 1D09C3 in early- or advanced-stage tumor xenografts. In vitro, 1D09C3-induced cell death involves a cascade of events, including ROS increase, JNK activation, mitochondrial membrane depolarization, and AIF release from mitochondria. Inhibition of JNK activity significantly reduced 1D09C3-induced apoptosis, indicating that 1D09C3 activity involves activation of the kinase. In vivo, 1D09C3 induces long-term disease-free survival in a significant proportion of tumor-bearing mice treated at an early stage of disease. Treatment of mice bearing advanced-stage lymphoma results in a highly significant prolongation of survival. These data show that 1D09C3 (a) exerts a potent antitumor effect by activating ROS-dependent, JNK-driven cell death, (b) cures the great majority of mice treated at an early-stage of disease, and (c) significantly prolongs survival of mice with advanced-stage disease.