Technology transfer of an oil-in-water vaccine-adjuvant for strengthening pandemic influenza preparedness in Indonesia.

With the current enzootic circulation of highly pathogenic avian influenza viruses, the ability to increase global pandemic influenza vaccine production capacity is of paramount importance. This has been highlighted by, and is one of the main pillars of, the WHO Global Action Plan for Influenza Vaccines (GAP). Such capacity expansion is especially relevant in developing countries. The Vaccine Formulation Laboratory at University of Lausanne is engaged in the technology transfer of an antigen-sparing oil-in-water adjuvant in order to empower developing countries vaccine manufacturers to increase pandemic influenza vaccine capacity. In a one-year project funded by United States Department of Health and Human Services, the Vaccine Formulation Laboratory transferred the process know-how and associated equipment for the pilot-scale manufacturing of an oil-in-water adjuvant to Bio Farma, Indonesia's state-owned vaccine manufacturer, for subsequent formulation with H5N1 pandemic influenza vaccines. This paper describes the experience acquired and lessons learnt from this technology transfer project.

Current drug discovery strategies against arenavirus infections.

Arenaviruses are a large group of emerging viruses including several causative agents of severe hemorrhagic fevers with high mortality in man. Considering the number of people affected and the currently limited therapeutic options, novel efficacious therapeutics against arenaviruses are urgently needed. Over the past decade, significant advances in knowledge about the basic virology of arenaviruses have been accompanied by the development of novel therapeutics targeting different steps of the arenaviral life cycle. High-throughput, small-molecule screens identified potent and broadly active inhibitors of arenavirus entry that were instrumental for the dissection of unique features of arenavirus fusion. Novel inhibitors of arenavirus replication have been successfully tested in animal models and hold promise for application in humans. Late in the arenavirus life cycle, the proteolytic processing of the arenavirus envelope glycoprotein precursor and cellular factors critically involved virion assembly and budding provide further promising 'druggable' targets for novel therapeutics to combat human arenavirus infection.

Incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study.

BACKGROUND: The incidence and outcomes of respiratory viral infections in lung transplant recipients (LTR) are not well defined. The objective of this prospective study conducted from June 2008 to March 2011 was to characterise the incidence and outcomes of viral respiratory infections in LTR. METHODS: Patients were seen in three contexts: study-specific screenings covering all seasons; routine post-transplantation follow-up; and emergency visits. Nasopharyngeal specimens were collected systematically and bronchoalveolar lavage (BAL) was performed when clinically indicated. All specimens underwent testing with a wide panel of molecular assays targeting respiratory viruses. RESULTS: One hundred and twelve LTR had 903 encounters: 570 (63%) were screening visits, 124 (14%) were routine post-transplantation follow-up and 209 (23%) were emergency visits. Respiratory viruses were identified in 174 encounters, 34 of these via BAL. The incidence of infection was 0.83 per patient-year (95% CI 0.45 to 1.52). The viral infection rates upon screening, routine and emergency visits were 14%, 15% and 34%, respectively (p<0.001). Picornavirus was identified most frequently in nasopharyngeal (85/140; 60.7%) and BAL specimens (20/34; 59%). Asymptomatic viral carriage, mainly of picornaviruses, was found at 10% of screening visits. Infections were associated with transient lung function loss and high calcineurin inhibitor blood levels. The hospitalisation rate was 50% (95% CI 30% to 70.9%) for influenza and parainfluenza and 16.9% (95% CI 11.2% to 23.9%) for other viruses. Acute rejection was not associated with viral infection (OR 0.4, 95% CI 0.1 to 1.3). CONCLUSIONS: There is a high incidence of viral infection in LTR; asymptomatic carriage is rare. Viral infections contribute significantly to this population's respiratory symptomatology. No temporal association was observed between infection and acute rejection.

HIV-1 protease mutation 82M contributes to phenotypic resistance to protease inhibitors in subtype G.

OBJECTIVES: The purpose of this study was the qualitative and quantitative assessment of the in vitro effect of HIV-1 protease (PR) mutation 82M on replication capacity and susceptibility to the eight clinically available PR inhibitors (PIs).¦METHODS: The 82M substitution was introduced by site-directed mutagenesis in wild-type subtype B and G strains, as well as reverted back to wild-type in a therapy-failing strain. The recombinant viruses were evaluated for their replication capacity and susceptibility to PIs.¦RESULTS: The single 82M mutation within a wild-type subtype B or G background did not result in drug resistance. However, the in vitro effect of single PR mutations on PI susceptibility is not always distinguishable from wild-type virus, and particular background mutations and polymorphisms are required to detect significant differences in the drug susceptibility profile. Consequently, reverting the 82M mutation back to wild-type (82I) in a subtype G isolate from a patient that failed therapy with multiple other PR mutations did result in significant increases in susceptibility towards indinavir and lopinavir and minor increases in susceptibility towards amprenavir and atazanavir. The presence of the 82M mutation also slightly decreased viral replication, whether it was in the genetic background of subtype B or subtype G.¦CONCLUSIONS: Our results suggest that 82M has an impact on PI susceptibility and that this effect is not due to a compensatory effect on the replication capacity. Because 82M is not observed as a polymorphism in any subtype, these observations support the inclusion of 82M in drug resistance interpretation systems and PI mutation lists.

Disinfection with sodium hypochlorite in hospital environmental surfaces in the reduction of contamination and infection prevention: a systematic review

OBJECTIVETo search for evidence of the efficiency of sodium hypochlorite on environmental surfaces in reducing contamination and prevention of healthcare-associated infection HAIs.METHODSystematic review in accordance with the Cochrane Collaboration.RESULTSWe analyzed 14 studies, all controlled trials, published between 1989-2013. Most studies resulted in inhibition of microorganism growth. Some decreased infection, microorganism resistance and colonization, loss of efficiency in the presence of dirty and surface-dried viruses.CONCLUSIONThe hypochlorite is an effective disinfectant, however, the issue of the direct relation with the reduction of HAIs remains. The absence of control for confounding variables in the analyzed studies made the meta-analysis performance inadequate. The evaluation of internal validity using CONSORT and TREND was not possible because its contents were not appropriate to laboratory and microbiological studies. As a result, there is an urgent need for developing specific protocol for evaluating such studies.

Amoeba/amoebal symbiont genetic transfers: lessons from giant virus neighbours.

Free-living amoebae serve as hosts for a variety of amoebae-resisting microorganisms, including giant viruses and certain bacteria. The latter include symbiotic bacteria as well as bacteria exhibiting a pathogenic phenotype towards amoebae. Amoebae-resisting bacteria have been shown to be widespread in water and to use the amoebae as a reservoir, a replication niche, a protective armour as well as a training ground to select virulence traits allowing survival in the face of microbicidal effects of macrophages, the first line of defense against invading pathogens. More importantly, amoebae play a significant role as a melting pot for genetic exchanges. These ecological and evolutionary roles of amoebae might also be at play for giant viruses and knowledge derived from the study of amoebae-resisting bacteria is useful for the study and understanding of interactions between amoebae and giant viruses. This is especially important since some genes have spread in all domains of life and the exponential availability of eukaryotic genomes and metagenomic sequences will allow researchers to explore these genetic exchanges in a more comprehensive way, thus completely changing our perception of the evolutionary history of organisms. Thus, a large part of this review is dedicated to report current known gene exchanges between the different amoebae-resisting organisms and between amoebae and the internalized bacteria.

Endosymbiotic bacteria associated with nematodes, ticks and amoebae.

Endosymbiosis is a mutualistic, parasitic or commensal symbiosis in which one symbiont is living within the body of another organism. Such symbiotic relationship with free-living amoebae and arthropods has been reported with a large biodiversity of microorganisms, encompassing various bacterial clades and to a lesser extent some fungi and viruses. By contrast, current knowledge on symbionts of nematodes is still mainly restricted to Wolbachia and its interaction with filarial worms that lead to increased pathogenicity of the infected nematode. In this review article, we aim to highlight the main characteristics of symbionts in term of their ecology, host cell interactions, parasitism and co-evolution, in order to stimulate future research in a field that remains largely unexplored despite the availability of modern tools.

Encéphalites virales [Viral encephalitis: update]

Suspicion of viral encephalitis should always be considered as a medical emergency and the prognosis depend on both the immune status of the host and the virulence of the virus. Among them, the herpes simplex virus is by far the most important one since it can be associated with severe encephalitis in immunocompetent host, and because a good response to acyclovir can be expected when rapidly initiated. Nevertheless, confirmation of the diagnosis requires exclusion of both metabolic or toxic encephalopathy and inflammatory encephalitis of non-infectious origin. In addition, other germs than viruses can mimic viral encephalitis and must be taken into consideration. The purpose of this review is to update the investigation that should be performed in clinical practice for any patient with suspicion of acute viral encephalitis.

Biosafety of Recombinant Adeno-associated Virus Vectors.

It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soon become a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method will have to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expression to alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use in gene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigators in the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhance safety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors based upon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only known human DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratory and animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented, we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapy clinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasis on biosafety.

Molecular epidemiology and evolution of HIV-1 in Portugal and portuguese speaking african countries

The aims of this thesis were to better characterize HIV-1 diversity in Portugal, Angola, Mozambique and Cape Verde and to investigate the origin and epidemiological history of HIV-1 in these countries. The impact of these issues in diagnosis, disease progression and susceptibility to ARV therapy was also investigated. Finally, the nature, dynamics and prevalence of transmitted drug resistance (TDR) was determined in untreated HIV-1 infected patients. In Angola, practically all HIV-1 genetic forms were found, including almost all subtypes, untypable (U) strains, CRFs and URFs. Recombinants (first and second generation) were present in 47.1% of the patients. HIV/AIDS epidemic in Angola probably started in 1961, the major cause being the independence war, subsequently spreading to Portugal. In Maputo, 81% of the patients were infected with subtype C viruses. Subtype G, U and recombinants such as CRF37_cpx, were also present. The results suggest that HIV-1 epidemic in Mozambique is evolving rapidly in genetic complexity. In Cape Verde, where HIV-1 and HIV-2 co-circulate, subtype G is the prevailed subtype. Subtypes B, C, F1, U, CRF02_AG and other recombinant strains were also found. HIV-2 isolates belonged to group A, some being closely related to the original ROD isolate. In all three countries numerous new polymorphisms were identified in the RT and PR of HIV-1 viruses. Mutations conferring resistance to the NRTIs or NNRTIs were found in isolates from 2 (2%) patients from Angola, 4 (6%) from Mozambique and 3 (12%) from Cape Verde. None of the isolates containing TDR mutations would be fully sensitive to the standard first-line therapeutic regimens used in these countries. Close surveillance in treated and untreated populations will be crucial to prevent further transmission of drug resistant strains and maximize the efficacy of ARV therapy. In Portugal, investigation of a seronegative case infection with rapid progression to AIDS and death revealed that the patient was infected with a CRF14_BG-like R5-tropic strain selectively transmitted by his seropositive sexual partner. The results suggest a massive infection with a highly aggressive CRF14_BG like strain and/or the presence of an unidentified immunological problem that prevented the formation of HIV-1-specific antibodies. Near full-length genomic sequences obtained from three unrelated patients enabled the first molecular and phylogenomic characterization of CRF14_BG from Portugal; all sequences were strongly related with CRF14_BG Spanish isolates. The mean date of origin of CRF14_BG was estimated to be 1992. We propose that CRF14_BG emerged in Portugal in the early 1990s, spread to Spain in late 1990s as a consequence of IDUs migration and then to the rest of Europe. Most CRF14_BG strains were predicted to use CXCR4 and were associated with rapid CD4 depletion and disease progression. Finally, we provide evidence suggesting that the X4 tropism of CRF14_BG may have resulted from convergent evolution of the V3 loop possibly driven by an effective escape from neutralizing antibody response.

Co-infecção por vírus da hepatite B e vírus da imunodeficiência humana em países africanos

Introdução: Actualmente, estima-se que existam dois milhões de indivíduos infectados por vírus da hepatite B (VHB) e que, cerca de 25% dos indivíduos com infecção crónica morrem devido a sequelas resultantes da infecção por VHB. Paralelamente, calcula-se que existam cerca de 33 milhões de indivíduos infectados por VIH, sendo que 22, 5 milhões residem na região de África a sul do Sara. Na região de África a sul do Sara existem poucos estudos efectuados no âmbito da co-infecção por VIH/VHB. Contudo, dos estudos existentes, esta taxa pode situar-se entre os 2,4% e os 9,9%. Objectivo: Avaliar as taxas de seroprevalência de VHB e VIH, assim como a taxa de co-infecção por VIH/VHB em Angola, Cabo Verde, Guiné-Bissau e Moçambique. Métodos: Foram efectuadas duas pesquisas bibliográficas neste estudo. A primeira, realizada nos meses de Setembro/Outubro 2008, tinha como objectivo contextualizar a infecção por VHB, VIH e a co-infecção por VIH/VHB nos países desenvolvidos e nos países em desenvolvimento. A segunda pesquisa foi efectuada durante o mês de Agosto de 2009, e visava apenas cobrir a realidade dos países em análise, relativamente aos objectivos previamente delineados do estudo. Resultados: Em Moçambique, constatou-se que a seroprevalência de VIH-1 tinha quadriplicado entre 1993 (1,17%) e o ano 2000 (4,5%). Na Guiné-Bissau, entre 1997 e 1999, também a seroprevalência de VIH-1 duplicou (2,5% e 5,2%, respectivamente). Em Cabo-Verde, no ano de 2006, a seroprevalência de VIH era 2,4%, enquanto que a seroprevalência da infecção por VHB era 4,4%. Em Angola, no ano de 2005, a seroprevalência de VIH era de 2,5%. Neste estudo também foi avaliada a co-infecção, sendo que nenhum caso foi diagnosticado. Conclusão: É urgente realizarem-se mais estudos nos países PALOP, no âmbito da seroprevalência das monoinfecções VIH e VHB, assim como na co-infecção por VIH/VHB, uma vez que existe pouca informação disponível. De qualquer modo, sendo a infecção por VHB uma doença prevenível por vacina, é fundamental que os planos de vacinação continuem a ser postos em prática nos países onde já estão implementados e, no caso dos países que ainda não os têm, que a sua implementação seja efectuada de forma sustentada e o mais brevemente possível.

Differential Recognition of Old World and New World Arenavirus Envelope Glycoproteins by Subtilisin Kexin Isozyme 1 (SKI-1)/Site 1 Protease (S1P).

The arenaviruses are an important family of emerging viruses that includes several causative agents of severe hemorrhagic fevers in humans that represent serious public health problems. A crucial step of the arenavirus life cycle is maturation of the envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). Comparison of the currently known sequences of arenavirus GPCs revealed the presence of a highly conserved aromatic residue at position P7 relative to the SKI-1/S1P cleavage side in Old World and clade C New World arenaviruses but not in New World viruses of clades A and B or cellular substrates of SKI-1/S1P. Using a combination of molecular modeling and structure-function analysis, we found that residueY285 of SKI-1/S1P, distal from the catalytic triad, is implicated in the molecular recognition of the aromatic "signature residue" at P7 in the GPC of Old World Lassa virus. Using a quantitative biochemical approach, we show that Y285 of SKI-1/S1P is crucial for the efficient processing of peptides derived from Old World and clade C New World arenavirus GPCs but not of those from clade A and B New World arenavirus GPCs. The data suggest that during coevolution with their mammalian hosts, GPCs of Old World and clade C New World viruses expanded the molecular contacts with SKI-1/S1P beyond the classical four-amino-acid recognition sequences and currently occupy an extended binding pocket.

ViralZone: a knowledge resource to understand virus diversity.

The molecular diversity of viruses complicates the interpretation of viral genomic and proteomic data. To make sense of viral gene functions, investigators must be familiar with the virus host range, replication cycle and virion structure. Our aim is to provide a comprehensive resource bridging together textbook knowledge with genomic and proteomic sequences. ViralZone web resource (www.expasy.org/viralzone/) provides fact sheets on all known virus families/genera with easy access to sequence data. A selection of reference strains (RefStrain) provides annotated standards to circumvent the exponential increase of virus sequences. Moreover ViralZone offers a complete set of detailed and accurate virion pictures.

Analysis of antiviral immunity in primary and chronic infections

Several evidences in humans underscored the contribution of CD4 and CD8 T-cell responses in controlling viral and bacterial infections. However, CD4 and CD8 Τ cells have distinct and specific effector functions leading to a hierarchical importance in responding to different types of pathogens. In this context, the present work aimed to investigate distinct CD8 T-cell features potentially influencing T-cell efficacy against viral infection. To achieve this-objective, CD8 Τ cells derived from HIV-infected patients and healthy donors harbouring virus-specific immune responses or immunized with an HTV vaccine candidate were studied. In particular, we performed a comprehensive cross-sectional and longitudinal analysis to characterize the function, the phenotype and the functional avidity of HIV-specific CD8 Τ cells during acute (PHI) and chronic infection and, in particular, we investigated immunological parameters potentially associated with the functional avidity of HIV-specific CD8 Τ cells. In addition, we studied the expression pattern of co-inhibitory molecules and the influence of CD 160 on the functions of CD8 Τ cells in absence of chronic infections. From these analyses we observed that the functional avidity of HIV-specific CD8 T- cell responses was significantly lower in acute than in chronic infection, but was not different between chronic progressive and non-progressive patients. Functional avidity remained low after several years of antiretroviral therapy in PHI patients, but increased in patients experiencing a virus rebound following treatment interruption in association with a massive renewal of the global CD8 complementarity-determining region 3 of the TCR. The functional avidity was also directly associated to T-cell exhaustion. In individuals with no sign of HIV or Hepatitis A, Β or C virus infection, CD8 Τ cells expressed higher levels of co-inhibitory molecules than CD4 Τ cells and this was dependent on the stage of T-cell differentiation and activation. The expression of CD 160 impaired the proliferation capacity and IL-2 production of CD8 Τ cells and was reduced upon CD8 T-cell activation, entitling CD 160 as unique marker of CD8 T-cell exhaustion. The CD 160 blockade restored the proliferation capacity of virus-specific CD8 Τ cells providing a potential new target for immunotherapy. All together, these results expand our knowledge regarding the interplay between the immune system and the viruses. - De nombreuses études chez l'Homme ont mis en évidence la contribution des réponses cellulaires Τ CD4 et CD8 dans le contrôle des infections virales et bactériennes. En particulier, les lymphocytes Τ ont différentes fonctions effectrices spécifiques qui leur confèrent un rôle clé lors d'infections par différents pathogènes. Ce travail vise à étudier différentes caractéristiques des cellules Τ CD8 affectant l'efficacité des réponses cellulaires contre les virus. Pour atteindre cet objectif nous avons étudié les cellules Τ CD8 provenant de patients infectés par le VIH et de donneurs sains avec des réponses immunitaires naturelles ou vaccinales contre des virus. Nous avons effectué plusieurs analyses transversales et longitudinales des fonctions, du phénotype et de l'avidité fonctionnelle des lymphocytes Τ CD8 spécifiques au VIH au cours d'infections aiguës et chroniques; en particulier, nous avons étudié les paramètres immunologiques qui pourraient être associés à l'avidité fonctionnelle. De plus, nous avons investigué le profil d'expression des principales molécules co-inhibitrices et en particulier le rôle du CD 160 dans les fonctions des lymphocytes Τ CD8. Sur la base de ces analyses, nous avons constaté que l'avidité fonctionnelle des cellules Τ CD8 spécifiques au VIH était significativement plus faible lors infections aiguës que lors d'infections chroniques, mais n'était, par contre, pas différente entre les patients avec des infections chroniques progressives et non progressives. L'avidité fonctionnelle reste faible après plusieurs années de traitement antirétroviral, mais augmente chez les patients subissant un rebond viral, et donc exposés à des hautes virémies, suite à l'interruption du traitement. Cette augmentation d'avidité des lymphocytes Τ CD8, liée à un épuisement fonctionnel accru, était quantitativement directement associée à un renouvellement massif du TCR. Indépendamment de l'infection par le VIH, les cellules Τ CD8 expriment des niveaux plus élevés de molécules co-inhibitrices (PD-1, 2B4 et CD 160) par rapport aux cellules Τ CD4 et ceci dépend de leur stade de différenciation et d'activation. En particulier, CD 160 semble être un marqueur clé d'épuisement cellulaire des cellules Τ CD8, et donc une nouvelle cible potentielle pour l'immunothérapie, car a) son expression réduit la capacité proliférative et la production d'IL-2 b) CD 160 diminue suite à 1'activation et c) le blocage de CD 160 redonne la capacité proliférative aux cellules Τ CD8 spécifiques aux virus. - Le système immunitaire est un ensemble de cellules, tissus et organes indispensables pour limiter l'entrée des pathogènes à travers la peau et les muqueuses. Parmi les différentes cellules composant le système immunitaire, les cellules Τ CD4 et CD8 sont fondamentales pour le contrôle des infections virales et bactériennes. Les moyens pour combattre les différents pathogènes peuvent être cependant très variables. Les cellules Τ CD8, qui sont indispensables pour la lutte contre les virus, peuvent avoir différents niveaux de sensibilité; les cellules qui répondent à de faibles quantités d'antigène ont une forte sensibilité. Suite à une première infection virale, les cellules Τ CD8 ont une sensibilité plus faible que lors d'expositions répétées au même virus. En effet, la réexposition au pathogène induit une augmentation de sensibilité, grâce au recrutement et/ou à l'expansion de cellules Τ dotées d'une sensibilité plus élevée. Les cellules Τ CD8 avec une plus haute sensibilité semblent être caractérisées par une perte de fonctionnalité (épuisement fonctionnel associé à une haute expression de molécules dites inhibitrices). En absence d'infection, la fonction des molécules inhibitrices n'est pas encore clairement définie. Les cellules Τ CD8 montrent un niveau d'expression plus élevé de ces molécules par rapport aux cellules Τ CD4. Ceci dépend de l'état des cellules. Parmi ces molécules, le CD160 est associé à l'incapacité des cellules à proliférer et à produire de l'IL-2, une protéine importante pour la prolifération et la survie cellulaire. L'incapacité des cellules exprimant le CD 160 à proliférer en réponse à des virus peut être restaurée par le blocage fonctionnel du récepteur CD 160. Cette étude étoffe notre connaissance du rôle des cellules Τ CD8 ainsi que des conséquences induites par leur épuisement fonctionnel. Ces informations sont fondamentales pour le développement de nouvelles stratégies thérapeutiques et vaccinales.

Clinical microbiologists facing an anthrax alert.

Microbiological war and terrorist attacks are made to weaken populations by transmitting pathogenic and epidemic microorganisms. These bacteria or viruses are often difficult to diagnose. Anthrax alerts following September 2001 showed that most clinical microbiology laboratories were not adequately prepared, using obsolete diagnostic methods or being too slow to use accurate tools when facing a major threat. Following this period, most microbiology laboratories were prepared for bioterrorism alerts, in order to provide accurate and rapid results, although such events are rare and unexpected. In this review, we describe the organization and preparedness of our clinical microbiology laboratory regarding bioterrorism risk, although its main task is to perform routine diagnostic microbiology tests. To illustrate the difficulties, we briefly describe an anthrax alert.