Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus.

The importance of the vpr gene for simian immunodeficiency virus (SIV) replication, persistence, and disease progression was examined by using the infectious pathogenic molecular clone called SIVmac239. The ATG start codon of the vpr gene was converted to TTG by site-specific mutagenesis. The constructed Vpr- mutant virus is identical with the parental SIVmac239/nef-stop virus with the exception of this one nucleotide. These viruses replicated with similar kinetics and to similar extents in rhesus monkey lymphocyte cultures and in the human CEMX174 cell line. Five rhesus monkeys were inoculated with the Vpr- variant of SIVmac239/nef-stop, and two monkeys received SIVmac239/nef-stop as controls. Both controls showed reversion of the TAA stop signal in nef by 2 weeks postinfection, as has been observed previously. Reversion of the TAA stop codon in nef also occurred in the five monkeys that received the Vpr- variant, but reversion was delayed on average to about 4 weeks. Thus, the mutation in vpr appeared to delay the rapidity with which reversion occurred in the nef gene. Reversion of the TTG sequence in vpr to ATG was observed in three of the five test animals. Reversion in vpr was first observed in these three animals 4 to 8 weeks postinfection. No vpr revertants were found over the entire 66 weeks of observation in the other two test animals that received the vpr mutant. Antibodies to vpr developed in those three animals in which reversion of vpr was documented, but antibodies to vpr were not observed in the two animals in which reversion of vpr was not detected. Antibody responses to gag and to whole virus antigens were of similar strength in all seven animals. Both control animals and two of the test animals in which vpr reverted maintained high virus loads and developed progressive disease. Low virus burden and no disease have been observed in the two animals in which vpr did not revert and in the one animal in which vpr reversion was first detected only at 8 weeks. The reversion of vpr in three of the five test animals indicates that there is significant selective pressure for functional forms of vpr in vivo. Furthermore, the results suggest that both vpr and nef are important for maximal SIV replication and persistence in vivo and for disease progression.     

Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection

The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system.Avian H5N1 influenza viruses, currently circulating mainly in southeast Asia, are likely to cause the next influenza pandemic (18, 26, 37). The supply of embryonated eggs for traditional influenza vaccine production may be endangered in this case. Efforts to produce inactivated H5N1 vaccines in permanent cells have resulted in large-scale manufacturing, for instance, in Vero cells (21). This approach, based either on fermentation of H5N1 wild-type (wt) viruses (21) or on viruses attenuated by reverse genetics (9, 31), is the most straightforward strategy for egg-independent, rapid vaccine production.A further approach that may result in more widely available, egg-independent H5 vaccines is the use of recombinant viral vectors expressing protective antigens. Promising protection results were obtained so far with adenovirus-based vectors in mouse models (13, 14). Adenovirus vectors are usually produced in permanent complementing cell lines (11) and have been widely used in clinical trials. Cancellation of a recent trial involving human immunodeficiency virus adenovirus vectors due to suspected enhancement of disease, however, may complicate the future use of these vectors (41). Poxvirus vectors, including recombinant modified vaccinia virus Ankara (MVA) (1, 43), constitute a further class of vectors that have been used to express H5N1 influenza virus antigens (5, 22, 44, 46). Usually, however, the large-scale production of MVA is carried out in primary chicken cells, since these are the most efficient production substrates and are also accepted by regulators. In a pandemic, this production platform may not be available because permanent nontumorigenic avian cell lines are currently not available for production.In this study, we used a permanent cell line, modified Vero cells, to produce nonreplicating vaccinia virus vectors expressing the H5 hemagglutinin (HA), the major influenza virus protective antigen. The defective vaccinia virus (dVV) vectors are safe due to their lack of replication capacity in normal hosts, while they share the superior immunizing properties of poxviral live vaccines (15, 33). Previously, a permanent cell line based on rabbit kidney cells was engineered to express the essential vaccinia virus D4R gene encoding the enzyme uracil-DNA-glycosylase. This cell line allowed the construction of replication-deficient vaccinia virus vectors (15). In this work, a complementing system based on Vero cells was established and used to produce the defective vaccinia virus vector dVV-HA5. The vector was used to immunize mice and was compared to an inactivated whole-virus (whv) vaccine and to replicating control viruses. The dVV-HA5 candidate vaccine induced neutralizing antibodies and full protection, similar to results with an inactivated whv vaccine. Further, it is important to ensure that the immune responses generated by a pandemic influenza vaccine give long-lived, broad, cross-clade protection. While antibody responses to influenza virus provide protective immunity, T-cell responses are also thought to play an important role in clearance of and recovery from infections. Thus, a vaccine which can produce both effective humoral and T-cell responses would be advantageous. A vaccinia virus vector-based pandemic influenza vaccine has the potential to provide this advantage.     

Methylene blue for malaria in Africa: results from a dose-finding study in combination with chloroquine

Background

Atovaquone is part of the antimalarial drug combination atovaquone-proguanil (Malarone^®) and inhibits the cytochrome bc₁ complex of the electron transport chain in Plasmodium spp. Molecular modelling showed that amino acid mutations are clustered around a putative atovaquone-binding site resulting in a reduced binding affinity of atovaquone for plasmodial cytochrome b, thus resulting in drug resistance.

Methods

The prevalence of cytochrome b point mutations possibly conferring atovaquone resistance in Plasmodium falciparum isolates in atovaquone treatment-naïve patient cohorts from Lambaréné, Gabon and from South Western Ethiopia was assessed.

Results

Four/40 (10%) mutant types (four different single polymorphisms, one leading to an amino acid change from M to I in a single case) in Gabonese isolates, but all 141/141 isolates were wild type in Ethiopia were found.

Conclusion

In the absence of drug pressure, spontaneous and possibly resistance-conferring mutations are rare.     

A complement fixing polysaccharide from Biophytum petersianum Klotzsch, a medicinal plant from Mali, West Africa

Biophytum petersianum Klotzsch (syn. Biophytum sensitivum (L.) DC) is a medicinal plant having a traditional use, among others, as a wound healing remedy in Mali and other countries. As a water extract of the aerial parts of the plant is a frequently used preparation, we decided to look for a bioactive polysaccharide in this extract. One of the obtained polysaccharide fractions, BP100 III, isolated from a 100 degrees C water extract from the aerial parts of B. petersianum and having a monosaccharide composition typical for pectic substances, was shown to exhibit potent dose-dependent complement fixating activity. The BP100 III fraction was subjected to degradation by endo-alpha-d-(1-->4)-polygalacturonase, and three fractions were obtained by gel filtration. The highest molecular weight fraction, BP100 III.1, had a more potent activity in the complement test system than the native polymer, while the two lower molecular weight fractions were less active than the native polymer. The major part of BP100 III.1 consists of galacturonic acid and rhamnose, with branches being present on both the rhamnose and galacturonic acid residues. Arabinogalactan type II is also present in the polymer, indicating that BP100 III.1 has a structure typical of the hairy region of pectins. The major part of the two other fractions is a galacturonan, containing a strikingly high number of branch points, some to which xylose is attached. These results indicate that the pectic substance in B. petersianum contains both rhamnogalacturonan and xylogalacturonan regions.     

Crystal Structure of an Aquabirnavirus Particle: Insights into Antigenic Diversity and Virulence Determinism

Infectious pancreatic necrosis virus (IPNV), a pathogen of salmon and trout, imposes a severe toll on the aquaculture and sea farming industries. IPNV belongs to the Aquabirnavirus genus in the Birnaviridae family of bisegmented double-stranded RNA viruses. The virions are nonenveloped with a T=13l icosahedral capsid made by the coat protein VP2, the three-dimensional (3D) organization of which is known in detail for the family prototype, the infectious bursal disease virus (IBDV) of poultry. A salient feature of the birnavirus architecture is the presence of 260 trimeric spikes formed by VP2, projecting radially from the capsid. The spikes carry the principal antigenic sites as well as virulence and cell adaptation determinants. We report here the 3.4-Å resolution crystal structure of a subviral particle (SVP) of IPNV, containing 20 VP2 trimers organized with icosahedral symmetry. We show that, as expected, the SVPs have a very similar organization to the IBDV counterparts, with VP2 exhibiting the same overall 3D fold. However, the spikes are significantly different, displaying a more compact organization with tighter packing about the molecular 3-fold axis. Amino acids controlling virulence and cell culture adaptation cluster differently at the top of the spike, i.e., in a central bowl in IBDV and at the periphery in IPNV. In contrast, the spike base features an exposed groove, conserved across birnavirus genera, which contains an integrin-binding motif. Thus, in addition to revealing the viral antigenic determinants, the structure suggests that birnaviruses interact with different receptors for attachment and for cell internalization during entry.Birnaviruses form a distinct family of double-stranded RNA (dsRNA) viruses infecting vertebrates and invertebrates (18). Aquatic birnaviruses are the most abundant and diverse and are grouped in two separate genera: the Aquabirnavirus genus and the Blosnavirus genus, with infectious pancreatic necrosis virus (IPNV) and blotched snakehead virus (BSNV) (16) as respective type species. A third aquatic birnavirus of unassigned genus, Tellina virus 1, was recently described and found to be phylogenetically distant from the two established genera (40). However, the vast majority of aquatic birnaviruses are antigenically related to IPNV (i.e., belong to the Aquabirnavirus genus), regardless of host species or geographical origin (4, 11, 26, 39). They are implicated as etiological agents of disease in a variety of mollusks and fish species important in aquaculture, causing pathologies such as infectious pancreatic necrosis in salmonids, nephroblastoma and branchionephritis in eels, and gill necrosis in clams (21, 34, 50). Viruses in the Aquabirnavirus genus display considerable antigenic diversity and have substantial differences in biological properties such as host range and optimal replication temperature. These features contrast with the properties of other birnaviruses, in particular those infecting terrestrial species (avibirnaviruses and entomobirnaviruses). Based on reciprocal neutralization tests with polyclonal and monoclonal antibodies, nine cross-reacting serotypes (A1 to A9) have been defined for IPNV and related aquabirnaviruses (26). Serotype A2 (also known as Sp) is the most common serotype found in Europe.Birnavirus particles are nonenveloped, displaying a single-shelled T=13l icosahedral capsid of about 70 nm in diameter, composed of 260 trimers of viral protein 2 (VP2) (5, 15, 42). Internal to the virion are VP3, which forms a ribonucleoprotein complex with the genomic RNA (9, 27, 36), and VP1, the viral RNA-dependent RNA polymerase, which is found both free and covalently attached to the genomic RNA (19). The birnavirus genome consists of two segments of dsRNA. While the smaller segment B has a single open reading frame (ORF) coding for VP1, segment A has two, a large and a small ORF, encoding the polyprotein precursor pVP2-VP4-VP3 and the nonstructural VP5, respectively. VP4 is a protease that cleaves its own N and C termini off the polyprotein, thus also releasing pVP2 (the VP2 precursor) and VP3 within the infected cell (3, 20). Subsequent serial cleavages at the C terminus of pVP2 upon particle assembly yield mature VP2 (amino acids 1 to 442 of the IPNV polyprotein) and three other peptides that remain within the virion (22). The longest of these peptides was shown to destabilize cell membranes, suggesting a role during entry (40). Maturation of the pVP2 precursor during assembly and the presence of VP3 are important for correct morphogenesis of icosahedral, T=13l virus particles.Recombinant expression of mature VP2 alone leads to assembly of small, dodecahedral T=1 subviral particles (SVPs) containing 20 VP2 trimers (10). The crystal structures of the SVPs of infectious bursal disease virus of poultry (IBDV) were reported by several groups (15, 23, 31) as well as the three-dimensional (3D) structure of an intact T=13l IBDV virion (15).Exposed at the virion surface, VP2 displays the humoral antigenic determinants of the virus and is the only viral protein shown to induce protective immunity. Furthermore, VP2 plays a key role during virus entry, being responsible for receptor recognition (8). Key residues controlling virulence and cell culture adaptation of IPNV were thus found to map to VP2 (46).We report here the crystal structure of the IPNV SVP and show that, like its IBDV counterpart, it is composed of 20 VP2 trimers organized with T=1 icosahedral symmetry. IPNV VP2 displays the same fold, and the SVP is organized in the same way, as anticipated from the 43% overall amino acid sequence identity between the VP2s of the two viruses. The molecules differ significantly, however, in the loops of domain P, which forms the projections or spikes. In particular, the 3D structure shows a clustering of variable residues in the outermost loops at the top of domain P. Residues associated with virulence and cell culture adaptation map to the most peripheral of these loops—away from the 3-fold axis at the convex top of VP2. This is in contrast to IBDV, in which virulence determinants map to a central bowl at the top of the VP2 spike. These results provide new insights for understanding the determinism of antigenicity and virulence of Aquabirnavirus strains.     

Accuracy of Urine Circulating Cathodic Antigen Test for the Diagnosis of Schistosoma mansoni in Preschool-Aged Children before and after Treatment

Background

The Kato-Katz technique is widely used for the diagnosis of Schistosoma mansoni, but shows low sensitivity in light-intensity infections. We assessed the accuracy of a commercially available point-of-care circulating cathodic antigen (POC-CCA) cassette test for the diagnosis of S. mansoni in preschool-aged children before and after praziquantel administration.

Methodology

A 3-week longitudinal survey with a treatment intervention was conducted in Azaguié, south Côte d''Ivoire. Overall, 242 preschoolers (age range: 2 months to 5.5 years) submitted two stool and two urine samples before praziquantel administration, and 86 individuals were followed-up posttreatment. Stool samples were examined with duplicate Kato-Katz thick smears for S. mansoni. Urine samples were subjected to POC-CCA cassette test for S. mansoni, and a filtration method for S. haematobium diagnosis.

Principal Findings

Before treatment, the prevalence of S. mansoni, as determined by quadruplicate Kato-Katz, single CCA considering ‘trace’ as negative (t−), and single CCA with ‘trace’ as positive (t+), was 23.1%, 34.3% and 64.5%, respectively. Using the combined results (i.e., four Kato-Katz and duplicate CCA(t−)) as diagnostic ‘gold’ standard, the sensitivity of a single Kato-Katz, a single CCA(t−) or CCA(t+) was 28.3%, 69.7% and 89.1%, respectively. Three weeks posttreatment, the sensitivity of a single Kato-Katz, single CCA(t−) and CCA(t+) was 4.0%, 80.0% and 84.0%, respectively. The intensity of the POC-CCA test band reaction was correlated with S. mansoni egg burden (odds ratio = 1.2, p = 0.04).

Conclusions/Significance

A single POC-CCA cassette test appears to be more sensitive than multiple Kato-Katz thick smears for the diagnosis of S. mansoni in preschool-aged children before and after praziquantel administration. The POC-CCA cassette test can be recommended for the rapid identification of S. mansoni infections before treatment. Additional studies are warranted to determine the usefulness of POC-CCA for assessing drug efficacy and monitoring the impact of control interventions.     

Estimating true prevalence of Schistosoma mansoni from population summary measures based on the Kato-Katz diagnostic technique

BackgroundThe prevalence of Schistosoma mansoni infection is usually assessed by the Kato-Katz diagnostic technique. However, Kato-Katz thick smears have low sensitivity, especially for light infections. Egg count models fitted on individual level data can adjust for the infection intensity-dependent sensitivity and estimate the ‘true’ prevalence in a population. However, application of these models is complex and there is a need for adjustments that can be done without modeling expertise. This study provides estimates of the ‘true’ S. mansoni prevalence from population summary measures of observed prevalence and infection intensity using extensive simulations parametrized with data from different settings in sub-Saharan Africa.MethodologyAn individual-level egg count model was applied to Kato-Katz data to determine the S. mansoni infection intensity-dependent sensitivity for various sampling schemes. Observations in populations with varying forces of transmission were simulated, using standard assumptions about the distribution of worms and their mating behavior. Summary measures such as the geometric mean infection, arithmetic mean infection, and the observed prevalence of the simulations were calculated, and parametric statistical models fitted to the summary measures for each sampling scheme. For validation, the simulation-based estimates are compared with an observational dataset not used to inform the simulation.Principal findingsOverall, the sensitivity of Kato-Katz in a population varies according to the mean infection intensity. Using a parametric model, which takes into account different sampling schemes varying from single Kato-Katz to triplicate slides over three days, both geometric and arithmetic mean infection intensities improve estimation of sensitivity. The relation between observed and ‘true’ prevalence is remarkably linear and triplicate slides per day on three consecutive days ensure close to perfect sensitivity.Conclusions/significanceEstimation of ‘true’ S. mansoni prevalence is improved when taking into account geometric or arithmetic mean infection intensity in a population. We supply parametric functions and corresponding estimates of their parameters to calculate the ‘true’ prevalence for sampling schemes up to 3 days with triplicate Kato-Katz thick smears per day that allow estimation of the ‘true’ prevalence.     

Co-infection of Long-Term Carriers of Plasmodium falciparum with Schistosoma haematobium Enhances Protection from Febrile Malaria: A Prospective Cohort Study in Mali

Background

Malaria and schistosomiasis often overlap in tropical and subtropical countries and impose tremendous disease burdens; however, the extent to which schistosomiasis modifies the risk of febrile malaria remains unclear.

Methods

We evaluated the effect of baseline S. haematobium mono-infection, baseline P. falciparum mono-infection, and co-infection with both parasites on the risk of febrile malaria in a prospective cohort study of 616 children and adults living in Kalifabougou, Mali. Individuals with S. haematobium were treated with praziquantel within 6 weeks of enrollment. Malaria episodes were detected by weekly physical examination and self-referral for 7 months. The primary outcome was time to first or only malaria episode defined as fever (≥37.5°C) and parasitemia (≥2500 asexual parasites/µl). Secondary definitions of malaria using different parasite densities were also explored.

Results

After adjusting for age, anemia status, sickle cell trait, distance from home to river, residence within a cluster of high S. haematobium transmission, and housing type, baseline P. falciparum mono-infection (n = 254) and co-infection (n = 39) were significantly associated with protection from febrile malaria by Cox regression (hazard ratios 0.71 and 0.44; P = 0.01 and 0.02; reference group: uninfected at baseline). Baseline S. haematobium mono-infection (n = 23) did not associate with malaria protection in the adjusted analysis, but this may be due to lack of statistical power. Anemia significantly interacted with co-infection (P = 0.009), and the malaria-protective effect of co-infection was strongest in non-anemic individuals. Co-infection was an independent negative predictor of lower parasite density at the first febrile malaria episode.

Conclusions

Co-infection with S. haematobium and P. falciparum is significantly associated with reduced risk of febrile malaria in long-term asymptomatic carriers of P. falciparum. Future studies are needed to determine whether co-infection induces immunomodulatory mechanisms that protect against febrile malaria or whether genetic, behavioral, or environmental factors not accounted for here explain these findings.     

Pre-clinical efficacy and safety of experimental vaccines based on non-replicating vaccinia vectors against yellow fever

Background

Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable.

Methodology/Principal Findings

A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10⁵ TCID₅₀. Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles.

Conclusions/Significance

The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.     

Accuracy of Urine Circulating Cathodic Antigen (CCA) Test for Schistosoma mansoni Diagnosis in Different Settings of C?te d'Ivoire

Background

Promising results have been reported for a urine circulating cathodic antigen (CCA) test for the diagnosis of Schistosoma mansoni. We assessed the accuracy of a commercially available CCA cassette test (designated CCA-A) and an experimental formulation (CCA-B) for S. mansoni diagnosis.

Methodology

We conducted a cross-sectional survey in three settings of Côte d''Ivoire: settings A and B are endemic for S. mansoni, whereas S. haematobium co-exists in setting C. Overall, 446 children, aged 8–12 years, submitted multiple stool and urine samples. For S. mansoni diagnosis, stool samples were examined with triplicate Kato-Katz, whereas urine samples were tested with CCA-A. The first stool and urine samples were additionally subjected to an ether-concentration technique and CCA-B, respectively. Urine samples were examined for S. haematobium using a filtration method, and for microhematuria using Hemastix dipsticks.

Principal Findings

Considering nine Kato-Katz as diagnostic ‘gold’ standard, the prevalence of S. mansoni in setting A, B and C was 32.9%, 53.1% and 91.8%, respectively. The sensitivity of triplicate Kato-Katz from the first stool and a single CCA-A test was 47.9% and 56.3% (setting A), 73.9% and 69.6% (setting B), and 94.2% and 89.6% (setting C). The respective sensitivity of a single CCA-B was 10.4%, 29.9% and 75.0%. The ether-concentration technique showed a low sensitivity for S. mansoni diagnosis (8.3–41.0%). The specificity of CCA-A was moderate (76.9–84.2%); CCA-B was high (96.7–100%). The likelihood of a CCA-A color reaction increased with higher S. mansoni fecal egg counts (odds ratio: 1.07, p<0.001). A concurrent S. haematobium infection or the presence of microhematuria did not influence the CCA-A test results for S. mansoni diagnosis.

Conclusion/Significance

CCA-A showed similar sensitivity than triplicate Kato-Katz for S. mansoni diagnosis with no cross-reactivity to S. haematobium and microhematuria. The low sensitivity of CCA-B in our study area precludes its use for S. mansoni diagnosis.