A synthetic biology approach for a vaccine platform against known and newly emerging serotypes of bluetongue virus.

UNLABELLED: Bluetongue is one of the major infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arbovirus existing in nature in at least 26 distinct serotypes. Here, we describe the development of a vaccine platform for BTV. The advent of synthetic biology approaches and the development of reverse genetics systems has allowed the rapid and reliable design and production of pathogen genomes which can be subsequently manipulated for vaccine production. We describe BTV vaccines based on "synthetic" viruses in which the outer core proteins of different BTV serotypes are incorporated into a common tissue-culture-adapted backbone. As a means of validation for this approach, we selected two BTV-8 synthetic reassortants and demonstrated their ability to protect sheep against virulent BTV-8 challenge. In addition to further highlight the possibilities of genome manipulation for vaccine production, we also designed and rescued a synthetic BTV chimera containing a VP2 protein, including regions derived from both BTV-1 and BTV-8. Interestingly, while the parental viruses were neutralized only by homologous antisera, the chimeric proteins could be neutralized by both BTV-1 and BTV-8 antisera. These data suggest that neutralizing epitopes are present in different areas of the BTV VP2 and likely "bivalent" strains eliciting neutralizing antibodies for multiple strains can be obtained. IMPORTANCE: Overall, this vaccine platform can significantly reduce the time taken from the identification of new BTV strains to the development and production of new vaccines, since the viral genomes of these viruses can be entirely synthesized in vitro. In addition, these vaccines can be brought quickly into the market because they alter the approach, but not the final product, of existing commercial products.

Performance of rapid tests for discrimination between HIV-1 and/or HIV-2 infections.

Major differences exist between HIV-1 and HIV-2 in terms of epidemiology, pathogenicity, sensitivity to antiretrovirals. Determining the type of HIV infecting a patient is essential for management. The aim of this study was to evaluate the ability of simple/rapid tests to differentiate between HIV-1 and/or HIV-2 infections. We analyzed 116 samples from patients infected with HIV-1 (n = 61), HIV-2 (n = 47), or HIV-1+HIV-2 (n = 8) at the chronic stage of infection. Each sample was tested with SD Bioline HIV-1/2 3.0, ImmunoFlow HIV1-HIV2, ImmunoFlow HIV1-HIV2 (WB), Genie III HIV-1/HIV-2, ImmunoComb HIV1&2 BiSpot. HIV-1, or HIV-2 single infection was identified with a sensitivity ranging from 90% to 100%. The ability to detect dual infection was less sensitive (12.5-100%). SD Bioline HIV-1/2 3.0, ImmunoFlow HIV1-HIV2, and Genie III were unable to detect HIV-1 group O infection in one, one and two cases, respectively. The specificity of detection of HIV-1, HIV-2, or HIV-1+HIV-2 antibodies differed greatly (36-100%). ImmunoComb BiSpot had the highest sensitivity values (99-100% for HIV-1, 98% for HIV-2, and 75-87.5% for dual infection) and specificity values (94-100% for HIV-1, 100% for HIV-2, and 97-100% for dual infection). In conclusion, this study showed that no single rapid test had a perfect sensitivity/specificity ratio, particularly in the case of the double infections.

Characteristics of patients recently infected with HIV-1 non-B subtypes in France: a nested study within the mandatory notification system for new HIV diagnoses.

The presence of HIV-1 non-B subtypes in Western Europe is commonly attributed to migration of individuals from non-European countries, but the possible role of domestic infections with non-B subtypes is not well investigated. The French mandatory anonymous reporting system for HIV is linked to a virological surveillance using assays for recent infection (<6 months) and serotyping. During the first semester of years 2007 to 2010, any sample corresponding to a non-B recent infection was analyzed by sequencing a 415-bp env region, followed by phylogenetic analysis and search for transmission clusters. Two hundred thirty-three recent HIV-1 infections with non-B variants were identified. They involved 5 subtypes and 7 circulating recombinant forms (CRFs). Ninety-two cases (39.5%) were due to heterosexual transmissions, of which 39 occurred in patients born in France. Eighty-five cases (36.5%) were identified in men having sex with men (MSM). Forty-three recent non-B infections (18.5%) segregated into 14 clusters, MSM being involved in 11 of them. Clustered transmission events included 2 to 7 cases per cluster. The largest cluster involved MSM infected by a CRF02_AG variant. In conclusion, we found that the spread of non-B subtypes in France occurs in individuals of French origin and that MSM are particularly involved in this dynamic.

Comparison of the local safety of two multi-component feline vaccines, adjuvanted (1 mL) versus non-adjuvanted at reduced volume (0.5 mL), using computed tomography imaging.

In 2020, a new 0.5 mL presentation of PUREVAX® RCP FeLV was registered and introduced in Europe. The objectives of this study were to investigate the local safety of this non-adjuvanted vaccine at reduced volume by classical methods (clinical examination, histopathology) and to evaluate the suitability of an alternative non-invasive methodology, the computed tomography (CT). For this purpose, the course of local reactions was assessed for 3 months after subcutaneous injection of PUREVAX® RCP FeLV 0.5 mL and compared to an adjuvanted vaccine, LEUCOFELIGEN® FeLV/RCP 1.0 mL. Injection site reactions consisted mainly of swelling reactions, which were more frequent, more pronounced and long-lasting in the adjuvanted vaccine group. Microscopically, in this group, moderate to severe inflammatory reactions were observed on day 7 (D7) and D21 post-injection and still present on D84, while mild inflammatory lesions were observed in the non-adjuvanted vaccine group only on D7 and D21. With the adjuvanted vaccine, inflamed areas were measurable by CT scan in all cats on D7 and D21, whereas they were detected only on D7 and only in 20 % of cats from the non-adjuvanted vaccine group. Besides the higher frequency, the mean inflamed volume was nearly 300 times larger in adjuvanted vaccine group on D7. Using different methodologies, the favorable safety profile of PUREVAX® RCP FeLV 0.5 mL was confirmed. Furthermore, the vaccine is aligned with current vaccination guidelines by inducing less inflammatory reactions, being adjuvant-free and injectable under a reduced volume, thus improving the convenience of administration in recommended sites (eg, legs). CT scan proved to be a suitable non-invasive method for the experimental follow-up of injection site reactions, yielding results consistent with clinical assessment and histopathology on D7 and D21. CT scan substantiated large differences between the investigated vaccines with a more prominent inflammatory reaction after injection of an adjuvanted vaccine.

Continuous spread of HIV-1 subtypes D and CRF01_AE in France from 2003 to 2009.

Among 61 and 35 patients who were infected in France by viruses of the rare clades D and CRF01_AE, respectively, approximately half of them originated from areas where HIV-1 is endemic, but the data showed that both clades have spread in the French indigenous population, particularly in men having sex with men (MSM).

Evaluation of safety and immunogenicity of feline vaccines with reduced volume.

A non adjuvanted vaccine against feline herpesvirus, feline calicivirus, feline panleucopenia and feline leukemia has been formulated in reduced volume (0.5 ml) with the same antigen content as the conventional 1 ml presentation. This paper reports studies evaluating the safety and the immunogenicity of this reduced volume vaccine in comparison with the conventional volume vaccine. The safety of both vaccines was evaluated in a small sized laboratory trial. It was further tested in a randomized controlled field trial on a total of 398 cats. Immediate and delayed local and systemic adverse events were monitored after vaccination. The immunogenicity of each vaccine was also checked by serological antibody responses against the vaccines antigens during the laboratory trial. These studies showed that the 0.5 ml vaccine was well tolerated in cats, inducing less local events, while keeping the same immunogenicity as the corresponding 1 ml vaccine. Reducing the volume of the vaccine is a way to improve the convenience of administration and to help following vaccination guidelines with the aim of reducing the incidence of adverse events following vaccination.

A nosocomial outbreak of feline calicivirus associated virulent systemic disease in France.

This report describes a nosocomial outbreak of feline calicivirus (FCV) associated virulent systemic disease (VSD) in a French veterinary teaching hospital in 2005. The outbreak started in March and resolved within 1 month. Signs, clinical course, clinicopathological findings and lesions were typical of FCV-induced VSD. FCV infection was confirmed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Among the eight infected cats, two had to be euthanased, three died, and three recovered after medical treatment. Virus could not be confined inside the animal hospital and on two occasions, students' own cats became infected. Subsequent genetic sequencing studies confirmed that the eight cats were infected with the same strain of virus, and that it was distinct from those involved in the US and UK outbreaks of VSD. Virulence and viral excretion patterns of the isolated strain were further characterised by experimental infection.

Rdl gene polymorphism and sequence analysis and relation to in vivo fipronil susceptibility in strains of the cat flea.

The gamma-amino butyric acid (GABA)-gated chloride ion channel in the insect central nervous system is the target of cyclodiene and phenylpyrazole insecticides. Resistance to dieldrin has been reported in several insect species and was associated with a point mutation (Ala285 to Ser substitution) in the M2 transmembrane domain of the GABA-gated chloride ion channel (the resistant to dieldrin [Rdl] gene). A partial Rdl gene sequence was reported previously in specimens of the cat flea, Ctenocephalides felis (Bouché). Because the presence of the Rdl gene mutation coincided with a reduction in susceptibility to fipronil in some insect species, it has been inferred that a similar association may exist in cat fleas. The Rdl gene sequence was evaluated in 20-50 fleas each from six cat flea strains shown previously to be fully susceptible to fipronil. Total DNA or RNA from fleas was extracted using a commercial kit, and the sequence encompassing the single nucleotide polymorphism (SNP) position Rdl was amplified by polymerase chain reaction (PCR) or reverse transcription-PCR. Amplification products were sequenced on both strands. All tested strains were homozygous for the mutant allele (T nucleotide at SNP position); amino acid sequencing demonstrated the Ala285 to Ser substitution. The results of this study indicated that the Rdl gene mutation was uniformly present as homozygous alleles in strains of fleas that have been shown to be fully susceptible to topically applied fipronil and that the efficacy of fipronil against cat fleas was not impacted by the Rdl gene mutation.

Multiple Correspondence Analysis on Amino Acid Properties within the Variable Region of the Capsid Protein Shows Differences between Classical and Virulent Systemic Feline Calicivirus Strains.

Feline calicivirus (FCV) is a widespread and highly prevalent pathogen of domestic cats, responsible for mild upper respiratory tract disease. Outbreaks of severe virulent systemic disease (VSD) associated with FCV infection have been reported worldwide. VSD FCV strains have a broader tropism and cause a systemic vascular compromise. Despite clear differences in the pathogenesis of VSD and oral respiratory infections, attempts to identify specific molecular markers of VSD strains on the major capsid protein VP1 have failed. Region E of VP1 is responsible for the interaction with the cell receptor Junctional Adhesion Molecule JAM-1 (FeJAM-1) and with VP2 minor capsid protein during the entry of the virus. We carried out an original analysis on the sequences from region E of VSD and classical strains. A Multiple Correspondence Analysis was performed on a Boolean matrix built by coding sequences on the basis of their amino acid properties. For the first time, this approach was able to differentiate VSD and classical FCV. Seven remarkable residue positions were shown to be statistically significant for pathotype differentiation, mainly located in the N-terminal hypervariable part of region E. As structural analysis suggested an interaction of these residues with FeJAM-1 or VP2, post-binding events, and specific conformational changes may explain the difference of pathogenesis between pathotypes.

Difficulties in diagnosing group o human immunodeficiency virus type 1 acute primary infection.

We report a rare case of acute human immunodeficiency virus (HIV) type 1 group O infection in a French Caucasian woman. Her sexual partner was secondarily diagnosed with HIV infection, and transmission was confirmed by phylogenetic analysis. The unequal performance of many of the serologic and molecular assays commercially available leads to delays in diagnosis and affects patient management.

Prevalence of HIV-2 and HIV-1 group O infections among new HIV diagnoses in France: 2003-2006.

French national surveillance of new HIV diagnoses included the collection of dried serum spots to identify HIV serotypes. Between January 2003 and June 2006, 10,184 new diagnoses were reported. The proportions of HIV-2 and HIV-1 group O infections were 1.8 and 0.1%, respectively. Most of these cases occurred in patients infected through heterosexual contact and originated from the corresponding endemic areas. Three cases of HIV-2 infections were reported in non-African men having sex with men.

HIV surveillance combining an assay for identification of very recent infection and phylogenetic analyses on dried spots.

BACKGROUND: Transmitted/founder viruses isolated at the early stage of infection are indicators of the variants that are spreading within a population. The French reporting system for new HIV diagnoses is linked to a virological surveillance using dried serum spots. METHODS: We combined an immunoassay for very recent infection (less than 31 days) to a phylogenetic analysis of transmitted/founder viruses and sociodemographic information to analyze the dynamics of the HIV-1 epidemic during a 3-year period. Bayesian coalescent-based methods were used to explore the temporal and spatial dynamics of the identified clusters. RESULTS: Of 17 010 dried serum spots collected, 549 very recent infections were identified for which both env sequences and sociodemographic data were available. Non-B transmitted/founder viruses were found in 196 cases (35.7%), belonging to six subtypes and seven circulating recombinant forms. Forty-three dyads/clusters were identified (range 2-11 cases), including 107 individuals (19.5%), mainly MSM. The largest cluster involved MSM infected by a CRF02_AG variant. Reconstruction of viral migrations across time suggests that Paris was the major hub of dissemination. CONCLUSION: The study shows the feasibility of the surveillance of the HIV epidemic using this methodology. The observation of actively growing spatiotemporal clusters allows identification of specific networks that may be targets for intervention.

Responses to Hypoxia and Endoplasmic Reticulum Stress Discriminate the Development of Vitreous and Floury Endosperms of Conventional Maize (Zea mays) Inbred Lines.

Major nutritional and agronomical issues relating to maize (Zea mays) grains depend on the vitreousness/hardness of its endosperm. To identify the corresponding molecular and cellular mechanisms, most studies have been conducted on opaque/floury mutants, and recently on Quality Protein Maize, a reversion of an opaque2 mutation by modifier genes. These mutant lines are far from conventional maize crops. Therefore, a dent and a flint inbred line were chosen for analysis of the transcriptome, amino acid, and sugar metabolites of developing central and peripheral endosperm that is, the forthcoming floury and vitreous regions of mature seeds, respectively. The results suggested that the formation of endosperm vitreousness is clearly associated with significant differences in the responses of the endosperm to hypoxia and endoplasmic reticulum stress. This occurs through a coordinated regulation of energy metabolism and storage protein (i.e., zein) biosynthesis during the grain-filling period. Indeed, genes involved in the glycolysis and tricarboxylic acid cycle are up-regulated in the periphery, while genes involved in alanine, sorbitol, and fermentative metabolisms are up-regulated in the endosperm center. This spatial metabolic regulation allows the production of ATP needed for the significant zein synthesis that occurs at the endosperm periphery; this finding agrees with the zein-decreasing gradient previously observed from the sub-aleurone layer to the endosperm center. The massive synthesis of proteins transiting through endoplasmic reticulum elicits the unfolded protein responses, as indicated by the splicing of bZip60 transcription factor. This splicing is relatively higher at the center of the endosperm than at its periphery. The biological responses associated with this developmental stress, which control the starch/protein balance, leading ultimately to the formation of the vitreous and floury regions of mature endosperm, are discussed.

Antibodies to conserved epitopes of the HIV-1 envelope in sera from long-term non-progressors: prevalence and association with neutralizing activity.

OBJECTIVE: Previous studies have shown that broadly neutralizing antibodies (NAb) are more frequent in long-term non-progressors (LTNP) than in other HIV-1 infected patients, but nothing is known about the envelope regions targeted by these broadly NAb. We investigated whether the breadth of neutralizing activity of sera was associated with the presence of specific antibodies (2F5- and/or 4E10-like, b12-like or 2G12-like antibodies) directed against conserved epitopes known to be involved in broad neutralization. METHODS: We assessed the ability of sera from 67 LTNP of the French ANRS cohort (ANRS CO15) to neutralize four heterologous primary isolates of four various clades. Competitive and non-competitive ELISA were developed for the specific comparison of levels of antibodies against these specific epitopes in neutralizing and non-neutralizing sera from LTNP. RESULTS: We found that higher 2G12-like antibody levels were significantly associated with the broadest neutralizing activity in sera from LTNP. Levels of 2G12-like antibodies were higher in the sera that neutralized the four isolates than in the others, with a median of 5.7 microg/ml [interquartile range (IQR), 2.7-9.3 microg/ml] versus 2.3 microg/ml (IQR, 1.1-3.9 microg/ml) (Mann-Whitney test, P = 0.03). Levels of antibodies against the other targeted envelope epitopes did not differ significantly between broadly and non-broadly neutralizing sera. CONCLUSION: These results suggest that the antigenicity of the "silent face" of gp120 that exposes the 2G12 epitope should be analysed in more detail, to find ways to induce broadly neutralizing antibodies.

Immunisation with a combination of two complementary feline calicivirus strains induces a broad cross-protection against heterologous challenges.

Feline calicivirus (FCV) is characterised by a high degree of antigenic variation potentially compromising vaccine efficacy. Inclusion of several FCV strains or antigens in current vaccines could be a means to improve protection against antigenically distinct isolates. This study evaluated the synergy between two FCV strains (FCVG1 and FCV431) by comparing immunity induced by either strain with that provided by a combination of the two strains against an heterologous challenge with antigenically distant FCV strains (FCV393 and FCV220). Thirty-two SPF kittens were randomly allocated to four groups of eight cats in each group. Groups B, C and D cats were vaccinated once subcutaneously with strains FCVG1, FCV431, and FCVG1 + FCV431, respectively. Each kitten received a total dose of 10(3.4) CCID50 of FCV. Control group A was not immunised. On day 31, four cats from each group were challenged oronasally with FCV220 and four cats with FCV393. Following challenge, the cats were monitored for clinical signs, viral shedding and antibody responses. FCV220 and FCV393 induced severe clinical signs in control cats typical of FCV infection. Immunisation with both strains mixed together induced higher neutralizing antibody titres against FCV220 and FCV393 strains on average. Protection was observed in all groups, however combination of the two strains resulted in a better clinical protection and reduction of virus shedding after heterologous challenge. A moderate correlation was observed between neutralizing antibody titres at the time of challenge and protection against clinical signs. These results indicated that vaccines combining antigens from different FCV strains may induce a broader heterologous protection.

Canine herpesvirus-1 (CHV-1): clinical, serological and virological patterns in breeding colonies.

Canine herpesvirus-1 (CHV-1) is presumed to be enzootic in the dog population and is associated with reproductive disorders and neonatal mortality. To advise dog breeders towards an effective management of CHV-1 infected colonies, 27 breeding bitches were studied during one reproductive cycle in field conditions: the effect of cycle stage, kennel size, initial antibody titre, mating and gestation on serologic and viral excretion patterns was evaluated, while the association between reproductive disorders and CHV-1 antibody titres and viral excretion was also analysed. All initially seronegative bitches seroconverted, while 40% of the initially seropositive bitches became seronegative at one or two occasions. No difference in antibody patterns was observed between mated and unmated bitches. Of the mated bitches, 46% experienced infertility, foetal resorption or mummification. No difference in antibody patterns was observed depending on the occurrence of reproductive disorders even if a decrease in antibody titres during early or late-di-oestrus was often present. Significantly higher titres were observed at all cycle stages in large kennels. None of the vaginal and nasal samples or buffy coats tested positive for CHV-1 DNA. The mixed image of clinical and sub-clinical carriage in this study demonstrated CHV-1 has a complex and difficult to predict clinical behavior. Preventive management with vaccination of reproducing bitches in kennels with reproductive disorders should therefore be advised.

Lipid partitioning in maize (Zea mays L.) endosperm highlights relationships among starch lipids, amylose, and vitreousness.

Content and composition of maize endosperm lipids and their partition in the floury and vitreous regions were determined for a set of inbred lines. Neutral lipids, i.e., triglycerides and free fatty acids, accounted for more than 80% of endosperm lipids and are almost 2 times higher in the floury than in the vitreous regions. The composition of endosperm lipids, including their fatty acid unsaturation levels, as well as their distribution may be related to metabolic specificities of the floury and vitreous regions in carbon and nitrogen storage and to the management of stress responses during endosperm cell development. Remarkably, the highest contents of starch lipids were observed systematically within the vitreous endosperm. These high amounts of starch lipids were mainly due to lysophosphatidylcholine and were tightly linked to the highest amylose content. Consequently, the formation of amylose-lysophosphatidylcholine complexes has to be considered as an outstanding mechanism affecting endosperm vitreousness.

Diversity of trends of viremia and T-cell markers in experimental acute feline immunodeficiency virus infection.

OBJECTIVE: The early events of human immunodeficiency virus infection seem critical for progression toward disease and antiretroviral therapy initiation. We wanted to clarify some still unknown prognostic relationships between inoculum size and changes in various immunological and virological markers. Feline immunodeficiency virus infection could be a helpful model. METHODS: Viremia and T-cell markers (number of CD4, CD8, CD8ß(low)CD62L(neg) T-cells, CD4/CD8 ratio, and percentage of CD8ß(low)CD62L(neg) cells among CD8 T-cells) were measured over 12 weeks in 102 cats infected with different feline immunodeficiency virus strains and doses. Viremia and T-cell markers trajectory groups were determined and the dose-response relationships between inoculum titres and trajectory groups investigated. RESULTS: Cats given the same inoculum showed different patterns of changes in viremia and T-cell markers. A statistically significant positive dose-response relationship was observed between inoculum titre and i) viremia trajectory-groups (r = 0.80, p<0.01), ii) CD8ß(low)CD62L(neg) cell-fraction trajectory-groups (r = 0.56, p<0.01). Significant correlations were also found between viremia and the CD4/CD8 ratio and between seven out of ten T-cell markers. CONCLUSIONS: In cats, the infectious dose determines early kinetics of viremia and initial CD8+ T-cell activation. An expansion of the CD8ß(low)CD62L(neg) T-cells might be an early predictor of progression toward disease. The same might be expected in humans but needs confirmation.

Decreased specificity of an assay for recent infection in HIV-1-infected patients on highly active antiretroviral treatment: implications for incidence estimates.

The aim of this study was to estimate the rate of misclassification in treated HIV patients who initiated treatment at the chronic stage of HIV infection using an enzyme immunoassay (EIA) that discriminates between recent infection (RI; within 6 months) and established infection. The performance of EIA-RI was evaluated in 96 HIV-1 chronically infected patients on highly active antiretroviral therapy (HAART) with an undetectable viral load (VL) for at least 3 years. Demographic data, HIV-1 viral load, CD4(+) T-cell count, viral subtype, and treatment duration were collected. The subset of misclassified patients was further analyzed using samples collected annually. The impact on incidence estimates was evaluated by simulation. The specificity in treated patients was significantly lower (70.8 to 77.1%) than that observed in untreated patients (93.3 to 99.3%, P < 0.001). Patients falsely classified as recently infected had been treated for a longer period and had longer-term viral suppression than those correctly classified. The loss of specificity of the test due to treatment may have a dramatic impact on the accuracy of the incidence estimates, with a major impact when HIV prevalence is high. The cross-sectional studies intended to derive HIV incidence must collect information on treatment or, alternatively, should include detection of antiretroviral drugs in blood specimens to rule out treated patients from the calculations.