Rapid host adaptation by extensive recombination

Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination that should be applicable to any recombinogenic viruses amenable to the production of synthetic infectious genomes. Our approach is based on differences in fitness that generally exist between synthetic chimaeric genomes and the wild-type viruses from which they are constructed. In mixed infections of defective reciprocal chimaeras, selection strongly favours recombinant progeny genomes that recover a portion of wild-type fitness. Characterizing these evolved progeny viruses can highlight both important genetic fitness determinants and the contribution that recombination makes to the evolution of their natural relatives. Moreover, these experiments supply precise information about the frequency and distribution of recombination breakpoints, which can shed light on the mechanistic processes underlying recombination. We demonstrate the value of this approach using the small single-stranded DNA geminivirus, maize streak virus (MSV). Our results show that adaptive recombination in this virus is extremely efficient and can yield complex progeny genomes comprising up to 18 recombination breakpoints. The patterns of recombination that we observe strongly imply that the mechanistic processes underlying rolling circle replication are the prime determinants of recombination breakpoint distributions found in MSV genomes sampled from nature. © 2009 SGM.

Human immunodeficiency virus type 1 subtype C Gag virus-like particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice

Human immunodeficiency virus type 1 (HIV-1) subtype C is the predominant HIV in southern Africa, and is the target of a number of recent vaccine candidates. It has been proposed that a heterologous prime/boost vaccination strategy may result in stronger, broader and more prolonged immune responses. Since HIV-1 Gag Pr55 polyprotein can assemble into virus-like particles (VLPs) which have been shown to induce a strong cellular immune response in animals, we showed that a typical southern African subtype C Pr55 protein expressed in insect cells via recombinant baculovirus could form VLPs. We then used the baculovirus-produced VLPs as a boost to a subtype C HIV-1 gag DNA prime vaccination in mice. This study shows that a low dose of HIV-1 subtype C Gag VLPs can significantly boost the immune response to a single subtype C gag DNA inoculation in mice. These results suggest a possible vaccination regimen for humans. © 2004 SGM.

Strategies for the prevention of cervical cancer by human papillomavirus vaccination

As cervical cancer is causally associated with 14 high-risk types of human papillomavirus (HPV), a successful HPV vaccine will have a major impact on this disease. Although some persistent HPV infections progress to cervical cancer, host immunity is generally able to clear most HPV infections. Both cell-mediated and antibody responses have been implicated in influencing the susceptibility, persistence or clearance of genital HPV infection. There have been two clinical trials that show that vaccines based on virus-like particles (VLPs) made from the major capsid protein, L1, are able to type specifically protect against cervical intra-epithelial neoplasia and infection. However, there is no evidence that even a mixed VLP vaccine will protect against types not included in the vaccine, and a major challenge that remains is how to engineer protection across a broader spectrum of viruses. Strategies for production of HPV vaccines using different vaccine vectors and different production systems are also reviewed. © 2005 Elsevier Ltd. All rights reserved.

A prime-boost immunisation regimen using recombinant BCG and Pr55 gag virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons

Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South African HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55 gag virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses. These responses were predominantly CD8+ T cell-mediated and recognised similar epitopes as those targeted by humans with early HIV-1 subtype C infection. In addition, a Gag-specific humoral response was elicited. These data support the development of HIV-1 vaccines based on rBCG and Pr55 gag VLPs. © 2009 Elsevier Ltd. All rights reserved.

More men than women make mucosal IgA antibodies to human papillomavirus type 16 (HPV-16) and HPV-18: A study of oral HPV and oral HPV antibodies in a normal healthy population

Background: We have previously shown the high prevalence of oral anti-human papillomavirus type 16 (HPV-16) antibodies in women with HPV-associated cervical neoplasia. It was postulated that the HPV antibodies were initiated after HPV antigenic stimulation at the cervix via the common mucosal immune system. The present study aimed to further evaluate the effectiveness of oral fluid testing for detecting the mucosal humoral response to HPV infection and to advance our limited understanding of the immune response to HPV. Methods: The prevalence of oral HPV infection and oral antibodies to HPV types 16, 18 and 11 was determined in a normal, healthy population of children, adolescents and adults, both male and female, attending a dental clinic. HPV types in buccal cells were determined by DNA sequencing. Oral fluid was collected from the gingival crevice of the mouth by the OraSure method. HPV-16, HPV-18 and HPV-11 antibodies in oral fluid were detected by virus-like particle-based enzyme-linked immunosorbent assay. As a reference group 44 women with cervical neoplasia were included in the study. Results: Oral HPV infection was h ighest in children (9/114, 7.9%), followed by adolescents (4/78, 5.1%), and lowest in normal adults (4/116, 3.5%). The predominant HPV type found was HPV-13 (7/22, 31.8%) followed by HPV-32 (5/22, 22.7%). The prevalence of oral antibodies to HPV-16, HPV-18 and HPV-11 was low in children and increased substantially in adolescents and normal adults. Oral HPV-16 IgA was significantly more prevalent in women with cervical neoplasia (30/44, 68.2%) than the women from the dental clinic (18/69, 26.1% P = 0.0001). Significantly more adult men than women displayed oral HPV-16 IgA (30/47 compared with 18/69, OR 5.0, 95% CI 2.09-12.1, P < 0.001) and HPV-18 IgA (17/47 compared with 13/69, OR 2.4, 95% CI 0.97-6.2, P = 0.04). Conclusion: The increased prevalence of oral HPV antibodies in adolescent individuals compared with children was attributed to the onset of sexual activity. The increased prevalence of oral anti-HPV IgA in men compared with women was noteworthy considering reportedly fewer men than women make serum antibodies, and warrants further investigation. © 2006 Marais et al; licensee BioMed Central Ltd.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

HIV-1 sub-type C chimaeric VLPs boost cellular immune responses in mice

Several approaches have been explored to eradicate HIV; however, a multigene vaccine appears to be the best option, given their proven potential to elicit broad, effective responses in animal models. The Pr55 Gagprotein is an excellent vaccine candidate in its own right, given that it can assemble into large, enveloped, virus-like particles (VLPs) which are highly immunogenic, and can moreover be used as a scaffold for the presentation of other large non-structural HIV antigens. In this study, we evaluated the potential of two novel chimaeric HIV-1 Pr55 Gag-based VLP constructs - C-terminal fusions with reverse transcriptase and a Tat::Nef fusion protein, designated GagRT and GagTN respectively - to enhance a cellular response in mice when used as boost components in two types of heterologous prime-boost vaccine strategies. A vaccine regimen consisting of a DNA prime and chimaeric HIV-1 VLP boosts in mice induced strong, broad cellular immune responses at an optimum dose of 100 ng VLPs. The enhanced cellular responses induced by the DNA prime-VLP boost were two- to three-fold greater than two DNA vaccinations. Moreover, a mixture of GagRT and GagTN VLPs also boosted antigen-specific CD8+ and CD4+ T-cell responses, while VLP vaccinations only induced predominantly robust Gag CD4+ T-cell responses. The results demonstrate the promising potential of these chimaeric VLPs as vaccine candidates against HIV-1. © 2010 Pillay et al; licensee BioMed Central Ltd.

The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

Background. One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1) and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1), an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results. The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap) alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions. We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used. © 2011 Tanzer et al; licensee BioMed Central Ltd.

A method for systematically pooling data in very early stage construction price forecasting

Client owners usually need an estimate or forecast of their likely building costs in advance of detailed design in order to confirm the financial feasibility of their projects. Because of their timing in the project life cycle, these early stage forecasts are characterized by the minimal amount of information available concerning the new (target) project to the point that often only its size and type are known. One approach is to use the mean contract sum of a sample, or base group, of previous projects of a similar type and size to the project for which the estimate is needed. Bernoulli’s law of large numbers implies that this base group should be as large as possible. However, increasing the size of the base group inevitably involves including projects that are less and less similar to the target project. Deciding on the optimal number of base group projects is known as the homogeneity or pooling problem. A method of solving the homogeneity problem is described involving the use of closed form equations to compare three different sampling arrangements of previous projects for their simulated forecasting ability by a cross-validation method, where a series of targets are extracted, with replacement, from the groups and compared with the mean value of the projects in the base groups. The procedure is then demonstrated with 450 Hong Kong projects (with different project types: Residential, Commercial centre, Car parking, Social community centre, School, Office, Hotel, Industrial, University and Hospital) clustered into base groups according to their type and size.

Randomised controlled trial of an automated, interactive telephone intervention (TLC Diabetes) to improve type 2 diabetes management : Baseline findings and six-month outcomes

Background: Effective self-management of diabetes is essential for the reduction of diabetes-related complications, as global rates of diabetes escalate. Methods: Randomised controlled trial. Adults with type 2 diabetes (n = 120), with HbA1c greater than or equal to 7.5 %, were randomly allocated (4 × 4 block randomised block design) to receive an automated, interactive telephone-delivered management intervention or usual routine care. Baseline sociodemographic, behavioural and medical history data were collected by self-administered questionnaires and biological data were obtained during hospital appointments. Health-related quality of life (HRQL) was measured using the SF-36. Results: The mean age of participants was 57.4 (SD 8.3), 63 % of whom were male. There were no differences in demographic, socioeconomic and behavioural variables between the study arms at baseline. Over the six-month period from baseline, participants receiving the Australian TLC (Telephone-Linked Care) Diabetes program showed a 0.8 % decrease in geometric mean HbA1c from 8.7 % to 7.9 %, compared with a 0.2 % HbA1c reduction (8.9 % to 8.7 %) in the usual care arm (p = 0.002). There was also a significant improvement in mental HRQL, with a mean increase of 1.9 in the intervention arm, while the usual care arm decreased by 0.8 (p = 0.007). No significant improvements in physical HRQL were observed. Conclusions: These analyses indicate the efficacy of the Australian TLC Diabetes program with clinically significant post-intervention improvements in both glycaemic control and mental HRQL. These observed improvements, if supported and maintained by an ongoing program such as this, could significantly reduce diabetes-related complications in the longer term. Given the accessibility and feasibility of this kind of program, it has strong potential for providing effective, ongoing support to many individuals with diabetes in the future.

Comparing measures of fat-free mass in overweight older adults using three different bioelectrical impedance devices and three prediction equations

Objectives: To compare measures of fat-free mass (FFM) by three different bioelectrical impedance analysis (BIA) devices and to assess the agreement between three different equations validated in older adult and/or overweight populations. Design: Cross-sectional study. Setting: Orthopaedics ward of Brisbane public hospital, Australia. Participants: Twenty-two overweight, older Australians (72 yr ± 6.4, BMI 34 kg/m2 ± 5.5) with knee osteoarthritis. Measurements: Body composition was measured using three BIA devices: Tanita 300-GS (foot-to-foot), Impedimed DF50 (hand-to-foot) and Impedimed SFB7 (bioelectrical impedance spectroscopy (BIS)). Three equations for predicting FFM were selected based on their ability to be applied to an older adult and/ or overweight population. Impedance values were extracted from the hand-to-foot BIA device and included in the equations to estimate FFM. Results: The mean FFM measured by BIS (57.6 kg ± 9.1) differed significantly from those measured by foot-to-foot (54.6 kg ± 8.7) and hand-to-foot BIA (53.2 kg ± 10.5) (P < 0.001). The mean ± SD FFM predicted by three equations using raw data from hand-to-foot BIA were 54.7 kg ± 8.9, 54.7 kg ± 7.9 and 52.9 kg ± 11.05 respectively. These results did not differ from the FFM predicted by the hand-to-foot device (F = 2.66, P = 0.118). Conclusions: Our results suggest that foot-to-foot and hand-to-foot BIA may be used interchangeably in overweight older adults at the group level but due to the large limits of agreement may lead to unacceptable error in individuals. There was no difference between the three prediction equations however these results should be confirmed within a larger sample and against a reference standard.

Duration-dependant response of mixed-method pre-cooling for intermittent-sprint exercise in the heat

This study examined the effects of pre-cooling duration on performance and neuromuscular function for self-paced intermittent-sprint shuttle running in the heat. Eight male, team-sport athletes completed two 35-min bouts of intermittent-sprint shuttle running separated by a 15-min recovery on three separate occasions (33°C, 34% relative humidity). Mixed-method pre-cooling was completed for 20 min (COOL20), 10-min (COOL10) or no cooling (CONT) and reapplied for 5-min mid-exercise. Performance was assessed via sprint times, percentage decline and shuttle-running distance covered. Maximal voluntary contractions (MVC), voluntary activation (VA) and evoked twitch properties were recorded pre- and post-intervention and mid- and post-exercise. Core temperature (T c), skin temperature, heart rate, capillary blood metabolites, sweat losses, perceptual exertion and thermal stress were monitored throughout. Venous blood draws pre- and post-exercise were analyzed for muscle damage and inflammation markers. Shuttle-running distances covered were increased 5.2 ± 3.3% following COOL20 (P < 0.05), with no differences observed between COOL10 and CONT (P > 0.05). COOL20 aided in the maintenance of mid- and post-exercise MVC (P < 0.05; d > 0.80), despite no conditional differences in VA (P > 0.05). Pre-exercise T c was reduced by 0.15 ± 0.13°C with COOL20 (P < 0.05; d > 1.10), and remained lower throughout both COOL20 and COOL10 compared to CONT (P < 0.05; d > 0.80). Pre-cooling reduced sweat losses by 0.4 ± 0.3 kg (P < 0.02; d > 1.15), with COOL20 0.2 ± 0.4 kg less than COOL10 (P = 0.19; d = 1.01). Increased pre-cooling duration lowered physiological demands during exercise heat stress and facilitated the maintenance of self-paced intermittent-sprint performance in the heat. Importantly, the dose-response interaction of pre-cooling and sustained neuromuscular responses may explain the improved exercise performance in hot conditions.

Mixed-method pre-cooling reduces physiological demand without improving performance of medium-fast bowling in the heat

This study examined physiological and performance effects of pre-cooling on medium-fast bowling in the heat. Ten, medium-fast bowlers completed two randomised trials involving either cooling (mixed-methods) or control (no cooling) interventions before a 6-over bowling spell in 31.9±2.1°C and 63.5±9.3% relative humidity. Measures included bowling performance (ball speed, accuracy and run-up speeds), physical characteristics (global positioning system monitoring and counter-movement jump height), physiological (heart rate, core temperature, skin temperature and sweat loss), biochemical (serum concentrations of damage, stress and inflammation) and perceptual variables (perceived exertion and thermal sensation). Mean ball speed (114.5±7.1 vs. 114.1±7.2 km · h−1; P = 0.63; d = 0.09), accuracy (43.1±10.6 vs. 44.2±12.5 AU; P = 0.76; d = 0.14) and total run-up speed (19.1±4.1 vs. 19.3±3.8 km · h−1; P = 0.66; d = 0.06) did not differ between pre-cooling and control respectively; however 20-m sprint speed between overs was 5.9±7.3% greater at Over 4 after pre-cooling (P = 0.03; d = 0.75). Pre-cooling reduced skin temperature after the intervention period (P = 0.006; d = 2.28), core temperature and pre-over heart rates throughout (P = 0.01−0.04; d = 0.96−1.74) and sweat loss by 0.4±0.3 kg (P = 0.01; d = 0.34). Mean rating of perceived exertion and thermal sensation were lower during pre-cooling trials (P = 0.004−0.03; d = 0.77−3.13). Despite no observed improvement in bowling performance, pre-cooling maintained between-over sprint speeds and blunted physiological and perceptual demands to ease the thermoregulatory demands of medium-fast bowling in hot conditions.

Effects of mixed-method cooling on recovery of medium-fast bowling performance in hot conditions on consecutive days

This investigation examined physiological and performance effects of cooling on recovery of medium-fast bowlers in the heat. Eight, medium-fast bowlers completed two randomised trials, involving two sessions completed on consecutive days (Session 1: 10-overs and Session 2: 4-overs) in 31 ± 3°C and 55 ± 17% relative humidity. Recovery interventions were administered for 20 min (mixed-method cooling vs. control) after Session 1. Measures included bowling performance (ball speed, accuracy, run-up speeds), physical demands (global positioning system, counter-movement jump), physiological (heart rate, core temperature, skin temperature, sweat loss), biochemical (creatine kinase, C-reactive protein) and perceptual variables (perceived exertion, thermal sensation, muscle soreness). Mean ball speed was higher after cooling in Session 2 (118.9 ± 8.1 vs. 115.5 ± 8.6 km · h−1; P = 0.001; d = 0.67), reducing declines in ball speed between sessions (0.24 vs. −3.18 km · h−1; P = 0.03; d = 1.80). Large effects indicated higher accuracy in Session 2 after cooling (46.0 ± 11.2 vs. 39.4 ± 8.6 arbitrary units [AU]; P = 0.13; d = 0.93) without affecting total run-up speed (19.0 ± 3.1 vs. 19.0 ± 2.5 km · h−1; P = 0.97; d = 0.01). Cooling reduced core temperature, skin temperature and thermal sensation throughout the intervention (P = 0.001–0.05; d = 1.31–5.78) and attenuated creatine kinase (P = 0.04; d = 0.56) and muscle soreness at 24-h (P = 0.03; d = 2.05). Accordingly, mixed-method cooling can reduce thermal strain after a 10-over spell and improve markers of muscular damage and discomfort alongside maintained medium-fast bowling performance on consecutive days in hot conditions.