Effect of Human Papillomavirus (HPV) Education Intervention on HPV Knowledge and Awareness Among High School Learners in Eastern Cape Province of South Africa.

Little is known about the level of knowledge and awareness with regard to human papillomavirus (HPV) and its associated risks among adolescents and young adults in South Africa. A cross-sectional study was conducted to assess HPV infection and associated risks knowledge level among learners attending high schools in the Eastern Cape Province of South Africa. Learners (females and males) attending five selected schools in the Eastern Cape Province of South Africa participated. The intervention included knowledge pre-assessment, education through structured lecture, and post-education assessment. Self-administered questionnaires were used in both pre and post-intervention assessments. There were 2652 learners, who participated, with a median age of 18 years (IQR: 16-19). Female participants constituted 53.58% (1421/2652), and male participants were 46.42% (1231/2652). Before education intervention, only 4.08% (107/2623) of learners ever heard about HPV and 3.31% (87/2626) about HPV vaccination. Only 9.36% (247/2638) and 9.34% (246/2635) knew that HPV infection is sexually transmitted and associated with cervical cancer development, respectively. After education intervention, knowledge about HPV among learners increased significantly (p < 0.001). In post-education assessment, female high school learners were 66% more likely to acquire HPV knowledge than males (OR, 1.66; 95% CI, 1.40-1.97; p < 0.0001). Exposure to an educational intervention significantly increased learners' knowledge levels. The increasing burden of cervical cancer and other HPV-associated cancers are public health problems of concern. Therefore, the evaluation of educational interventions for increasing knowledge on HPV-associated diseases is necessary for low-resource settings with a high burden of cervical cancer.

Characterization of a dynamic self-replicating mammalian expression vector based on the circular ssDNA genome of beak and feather disease virus.

In vivo nucleic expression technologies using DNA or mRNA offer several advantages for recombinant gene expression. Their inherent ability to generate natively expressed recombinant proteins and antigens allows these technologies to mimic foreign gene expression without infection. Furthermore, foreign nucleic acid fragments have an inherent ability to act as natural immune adjuvants and stimulate innate pathogen- and DNA damage-associated receptors that are responsible for activating pathogen-associated molecular pattern (PAMP) and DNA damage-associated molecular pattern (DAMP) signalling pathways. This makes nucleic-acid-based expression technologies attractive for a wide range of vaccine and oncolytic immunotherapeutic uses. Recently, RNA vaccines have demonstrated their efficacy in generating strong humoral and cellular immune responses for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). DNA vaccines, which are more stable and easier to manufacture, generate similar immune responses to RNA, but typically exhibit lower immunogenicity. Here we report on a novel method of constructing self-amplifying DNA expression vectors that have the potential to amplify and enhance gene/antigen expression at a cellular level by increasing per cell gene copy numbers, boost genomic adjuvating effects and mitigate through replication many of the problems faced by non-replicating vectors such as degradation, methylation and gene silencing. These vectors employ a viral origin rolling circle replication cycle in mammalian host cells that amplifies the vector and gene of interest (GOI) copy number, maintaining themselves as nuclear episomes. We show that these vectors maintain persistently elevated GOI expression levels at the cellular level and induce morphological cellular alterations synonymous with increased cellular stress.

Augmenting glycosylation-directed folding pathways enhances the fidelity of HIV Env immunogen production in plants.

Heterologous glycoprotein production relies on host glycosylation-dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco-engineering to improve the production of a model HIV-1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3-fucosyltransferase and ß1,2-xylosyltransferase was used as an expression host to prevent plant-specific complex N-glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco-engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell-produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant-produced and mammalian cell-derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus-vectored vaccines. This study demonstrates that engineering glycosylation-directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

Development of a synthetic nanoparticle vaccine presenting the HIV-1 envelope glycoprotein.

Two-component self-assembling virus-like particles (VLPs) are promising scaffolds for achieving high-density display of HIV-1 envelope (gp140) trimers, which can improve the induction of neutralising antibodies (NAbs). In this study gp140 was displayed on the surface of VLPs formed by the AP205 phage coat protein. The CAP256 SU gp140 antigen was selected as the patient who this virus was isolated from developed broadly neutralising antibodies (bNAbs) shortly after superinfection with this virus. The CAP256 SU envelope is also sensitive to several bNAbs and has shown enhanced reactivity for certain bNAb precursors. A fusion protein comprising the HIV-1 CAP256 SU gp140 and the SpyTag (ST) (gp140-ST) was produced in HEK293 cells, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced inEscherichia coliand purified by ultracentrifugation. The gp140-ST trimers and the SC-AP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). Dynamic light scattering, negative stain electron microscopy and 2D classification indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs, and the second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens. The animals were then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. However, four inoculations of coupled VLPs alone failed to elicit any Tier 2 antibodies. These results demonstrate that the native-like structure of HIV-1 envelope trimers and selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of the trimers are important considerations that could improve the effect of nanoparticle-displayed gp140.

Influence of the lumpy skin disease virus (LSDV) superoxide dismutase homologue on host transcriptional activity, apoptosis and histopathology.

Lumpy skin disease virus (LSDV), a Capripoxvirus, is of economic importance in the cattle industry and is controlled by vaccination. A comparison was made of the host response to the two LSDV vaccines Neethling and Herbivac LS, with reference to the well-studied Orthopoxvirus, modified vaccinia Ankara (MVA), in a mouse model. Because the vaccines differ at the superoxide dismutase homologue (SOD) gene locus, recombinant SOD knock-out and knock-in nLSDV vaccines were constructed and all four vaccines were tested for the induction and inhibition of apoptosis. The SOD homologue was associated both with induction of apoptosis as well as inhibition of camptothecin-induced apoptosis. Histological analysis of chorioallantoic membranes of fertilized hens' eggs infected with the four different vaccines indicated marked mesodermal proliferation associated with vaccines containing the full-length SOD homologue as well as increased immune cell infiltration. Our findings suggest that the SOD homologue may influence vaccine immunogenicity.

Recombination Between High-Risk Human Papillomaviruses and Non-Human Primate Papillomaviruses: Evidence of Ancient Host Switching Among Alphapapillomaviruses.

We use all the currently known 405 Papillomavirus (PV) sequences, 343 curated PV sequences from both humans and animals from the PAVE data base, to analyse the recombination dynamics of these viruses at the whole genome levels. After showing some evidence of human and non-human primate PV recombination, we report a comprehensive recombination analysis of all currently known 82 Alphapapillomaviruses (Alpha-PVs). We carried out an exploratory study and found novel recombination events between High-Risk HPV Types and Macaca fascicularis PV1 (MfPV1), Macaca Fuscata PV2 (MfuPV2) and Pan Paniscus PV1 (PpPV1) Papillomaviruses. This is the first evidence of interactions between PVs from different hosts and hence postulates the likelihood of ancient host switching among Alpha-PVs. Notwithstanding these results should be interpreted with caution because the major and minor parents indicated by RDP4 program are simply the sequences in the alignment that most closely resemble the actual parents. We found statistically significant differences between the phylogenies of the PV sequences with recombination regions and PV sequences without recombination regions using the Shimodaira-Hasegawa phylogenetic incongruence testing. We show that not more than 76MYA Alpha-PVs were in the same biological niche, a pre-requisite for recombination, and as the hosts evolved and diversified, the viruses adapted to specific host niches which eventually led to coevolution with specific hosts before speciation of primate species. Thus providing evidence that in ancient times no earlier than the Cretaceous period of the Mesozoic age, Alpha-PVs recombined and switched hosts, but whether this host switching is occurring currently is unknown. However, a clearer picture of the PVs evolutionary landscape can only be achieved with the incremental discovery of PV sequences, especially from the animal kingdom.

Co-expression of human calreticulin significantly improves the production of HIV gp140 and other viral glycoproteins in plants.

Plant molecular farming (PMF) is rapidly gaining traction as a viable alternative to the currently accepted paradigm of producing biologics. While the platform is potentially cheaper and more scalable than conventional manufacturing systems, expression yields and appropriate post-translational modifications along the plant secretory pathway remain a challenge for certain proteins. Viral fusion glycoproteins in particular are often expressed at low yields in plants and, in some cases, may not be appropriately processed. Recently, however, transiently or stably engineering the host plant has shown promise as a strategy for producing heterologous proteins with more complex maturation requirements. In this study we investigated the co-expression of a suite of human chaperones to improve the production of a human immunodeficiency virus (HIV) type 1 soluble gp140 vaccine candidate in Nicotiana benthamiana plants. The co-expression of calreticulin (CRT) resulted in a dramatic increase in Env expression and ameliorated the endoplasmic reticulum (ER) stress response - as evidenced by lower transcript abundance of representative stress-responsive genes. The co-expression of CRT similarly improved accumulation of glycoproteins from Epstein-Barr virus (EBV), Rift Valley fever virus (RVFV) and chikungunya virus (CHIKV), suggesting that the endogenous chaperone machinery may impose a bottleneck for their production. We subsequently successfully combined the co-expression of human CRT with the transient expression of human furin, to enable the production of an appropriately cleaved HIV gp140 antigen. These transient plant host engineering strategies are a promising approach for the production of high yields of appropriately processed and cleaved viral glycoproteins.

Prime-Boost Immunizations with DNA, Modified Vaccinia Virus Ankara, and Protein-Based Vaccines Elicit Robust HIV-1 Tier 2 Neutralizing Antibodies against the CAP256 Superinfecting Virus.

A vaccine regimen that elicits broadly neutralizing antibodies (bNAbs) is a major goal in HIV-1 vaccine research. In this study, we assessed the immunogenicity of the CAP256 superinfecting viral envelope (CAP256 SU) protein delivered by modified vaccinia virus Ankara (MVA) and DNA vaccines in different prime-boost combinations followed by a soluble protein (P) boost. The envelope protein (Env) contained a flexible glycine linker and I559P mutation. Trimer-specific bNAbs PGT145, PG16, and CAP256 VRC26_08 efficiently bound to the membrane-bound CAP256 envelope expressed on the surface of cells transfected or infected with the DNA and MVA vaccines. The vaccines were tested in two different vaccination regimens in rabbits. Both regimens elicited autologous tier 2 neutralizing antibodies (NAbs) and high-titer binding antibodies to the matching CAP256 Env and CAP256 V1V2 loop scaffold. The immunogenicity of DNA and MVA vaccines expressing membrane-bound Env alone was compared to that of Env stabilized in a more native-like conformation on the surface of Gag virus-like particles (VLPs). The inclusion of Gag in the DNA and MVA vaccines resulted in earlier development of tier 2 NAbs for both vaccination regimens. In addition, a higher proportion of the rabbits primed with DNA and MVA vaccines that included Gag developed tier 2 NAbs than did those primed with vaccine expressing Env alone. Previously, these DNA and MVA vaccines expressing subtype C mosaic HIV-1 Gag were shown to elicit strong T cell responses in mice. Here we show that when the CAP256 SU envelope protein is included, these vaccines elicit autologous tier 2 NAbs.IMPORTANCE A vaccine is urgently needed to combat HIV-1, particularly in sub-Saharan Africa, which remains disproportionately affected by the AIDS pandemic and accounts for the majority of new infections and AIDS-related deaths. In this study, two different vaccination regimens were compared. Rabbits that received two DNA primes followed by two modified vaccinia virus Ankara (MVA) and two protein inoculations developed better immune responses than those that received two MVA and three protein inoculations. In addition, DNA and MVA vaccines that expressed mosaic Gag VLPs presenting a stabilized Env antigen elicited better responses than Env alone, which supports the inclusion of Gag VLPs in an HIV-1 vaccine.

Chronic schistosomiasis suppresses HIV-specific responses to DNA-MVA and MVA-gp140 Env vaccine regimens despite antihelminthic treatment and increases helminth-associated pathology in a mouse model.

Future HIV vaccines are expected to induce effective Th1 cell-mediated and Env-specific antibody responses that are necessary to offer protective immunity to HIV infection. However, HIV infections are highly prevalent in helminth endemic areas. Helminth infections induce polarised Th2 responses that may impair HIV vaccine-generated Th1 responses. In this study, we tested if Schistosoma mansoni (Sm) infection altered immune responses to SAAVI candidate HIV vaccines (DNA and MVA) and an HIV-1 gp140 Env protein vaccine (gp140) and whether parasite elimination by chemotherapy or the presence of Sm eggs (SmE) in the absence of active infection influenced the immunogenicity of these vaccines. In addition, we evaluated helminth-associated pathology in DNA and MVA vaccination groups. Mice were chronically infected with Sm and vaccinated with DNA+MVA in a prime+boost combination or MVA+gp140 in concurrent combination regimens. Some Sm-infected mice were treated with praziquantel (PZQ) prior to vaccinations. Other mice were inoculated with SmE before receiving vaccinations. Unvaccinated mice without Sm infection or SmE inoculation served as controls. HIV responses were evaluated in the blood and spleen while Sm-associated pathology was evaluated in the livers. Sm-infected mice had significantly lower magnitudes of HIV-specific cellular responses after vaccination with DNA+MVA or MVA+gp140 compared to uninfected control mice. Similarly, gp140 Env-specific antibody responses were significantly lower in vaccinated Sm-infected mice compared to controls. Treatment with PZQ partially restored cellular but not humoral immune responses in vaccinated Sm-infected mice. Gp140 Env-specific antibody responses were attenuated in mice that were inoculated with SmE compared to controls. Lastly, Sm-infected mice that were vaccinated with DNA+MVA displayed exacerbated liver pathology as indicated by larger granulomas and increased hepatosplenomegaly when compared with unvaccinated Sm-infected mice. This study shows that chronic schistosomiasis attenuates both HIV-specific T-cell and antibody responses and parasite elimination by chemotherapy may partially restore cellular but not antibody immunity, with additional data suggesting that the presence of SmE retained in the tissues after antihelminthic therapy contributes to lack of full immune restoration. Our data further suggest that helminthiasis may compromise HIV vaccine safety. Overall, these findings suggested a potential negative impact on future HIV vaccinations by helminthiasis in endemic areas.

The penile microbiota of Black South African men: relationship with human papillomavirus and HIV infection.

BACKGROUND: To date, the microbiota of the human penis has been studied mostly in connection with circumcision, HIV risk and female partner bacterial vaginosis (BV). These studies have shown that male circumcision reduces penile anaerobic bacteria, that greater abundance of penile anaerobic bacteria is correlated with increased cytokine levels and greater risk of HIV infection, and that the penile microbiota is an important harbour for BV-associated bacteria. While circumcision has been shown to significantly reduce the risk of acquiring human papillomavirus (HPV) infection, the relationship of the penile microbiota with HPV is still unknown. In this study, we examined the penile microbiota of HPV-infected men as well as the impact of HIV status. RESULTS: The penile skin microbiota of 238 men from Cape Town (South Africa) were profiled using Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. Corynebacterium and Prevotella were found to be the most abundant genera. Six distinct community state types (CSTs) were identified. CST-1, dominated by Corynebacterium, corresponded to less infections with high-risk HPV (HR-HPV) relative to CSTs 2-6. Men in CST-5 had greater relative abundances of Prevotella, Clostridiales, and Porphyromonas and a lower relative abundance of Corynebacterium. Moreover, they were significantly more likely to have HPV or HR-HPV infections than men in CST-1. Using a machine learning approach, we identified greater relative abundances of the anaerobic BV-associated bacteria (Prevotella, Peptinophilus, and Dialister) and lower relative abundance of Corynebacterium in HR-HPV-infected men compared to HR-HPV-uninfected men. No association was observed between HIV and CST, although the penile microbiota of HIV-infected men had greater relative abundances of Staphylococcus compared to HIV-uninfected men. CONCLUSIONS: We found significant differences in the penile microbiota composition of men with and without HPV and HIV infections. HIV and HR-HPV infections were strongly associated with greater relative abundances of Staphylococcus and BV-associated bacterial taxa (notably Prevotella, Peptinophilus and Dialister), respectively. It is possible that these taxa could increase susceptibility to HIV and HR-HPV acquisition, in addition to creating conditions in which infections persist. Further longitudinal studies are required to establish causal relationships and to determine the extent of the effect.

South African bovine ephemeral fever virus glycoprotein sequences are phylogenetically distinct from those from the rest of the world.

Bovine ephemeral fever virus (BEFV) is an economically important arbovirus affecting cattle and water buffalo. Currently, isolates can be separated into three phylogenetic groups, differentiated by the place of isolation, namely, East Asia, Australia, and the Middle East. BEFV surface glycoprotein (G) genes from 14 South African field strains collected between 1968 and 1999 were sequenced and compared to 154 published sequences. The BEFV isolates from South Africa were found to be phylogenetically distinct from those from other parts of the world.

Evolutionary dynamics of ten novel Gamma-PVs: insights from phylogenetic incongruence, recombination and phylodynamic analyses.

BACKGROUND: Human papillomaviruses (HPVs) are genetically diverse, belonging to five distinct genera: Alpha, Beta, Gamma, Mu and Nu. All papillomaviruses have double stranded DNA genomes that are thought to evolve slowly because they co-opt high-fidelity host cellular DNA polymerases for their replication. Despite extensive efforts to catalogue all the HPV species that infect humans, it is likely that many still remain undiscovered. Here we use the sequences of ten novel Gammapapillomaviruses (Gamma-PVs) characterized in previous studies and related HPVs to analyse the evolutionary dynamics of these viruses at the whole genome and individual gene scales. RESULTS: We found statistically significant incongruences between the phylogenetic trees of different genes which imply gene-to-gene variation in the evolutionary processes underlying the diversification of Gamma-PVs. We were, however, only able to detect convincing evidence of a single recombination event which, on its own, cannot explain the observed incongruences between gene phylogenies. The divergence times of the last common ancestor (LCA) of the Alpha, Beta, Mu, Nu and Gamma genera was predicted to have existed between 49.7-58.5 million years ago, before splitting into the five main lineages. The LCA of the Gamma-PVs at this time was predicted to have existed between 45.3 and 67.5 million years ago: approximately at the time when the simian and tarsier lineages of the primates diverged. CONCLUSION: Consequently, we report here phylogenetic tree incongruence without strong evidence of recombination.

Inflammatory cytokine biomarkers of asymptomatic sexually transmitted infections and vaginal dysbiosis: a multicentre validation study.

OBJECTIVES: Vaginal dysbiosis and STIs are important drivers of the HIV epidemic and reproductive complications. These conditions remain prevalent, partly because most cases are asymptomatic. We have shown that inflammatory cytokines interleukin (IL)-1α, IL-1ß and interferon-γ-induced protein (IP)-10 are biomarkers for detecting asymptomatic STIs and vaginal dysbiosis (bacterial vaginosis (BV) or intermediate microbiota). This study aimed to validate the performance of these biomarkers in African women recruited regardless of symptoms. METHODS: IL-1α, IL-1ß and IP-10 were measured in menstrual cup secretions, endocervical, lateral vaginal wall and vulvovaginal swabs from 550 women from Pretoria, Soweto and Cape Town, South Africa and Bondo, Kenya using Luminex and ELISA. STIs were assessed by PCR, BV by Nugent scoring and vaginal microbiota by 16S rRNA sequencing. RESULTS: Across four study populations and four types of genital specimens, the performance of IL-1α, IL-1ß and IP-10 for identification of women with STIs, BV or intermediate microbiota was consistent. Of the genital samples assessed, biomarkers measured in lateral vaginal wall swabs performed best, correctly classifying 76%(95% CI 70% to 81%) of women according to STI, BV or intermediate microbiota status (sensitivity 77%, specificity 71%) and were more accurate than clinical symptoms (sensitivity 41%, specificity 57%) (p=0.0003). Women incorrectly classified as STI/BV positive using the biomarkers had more abundant dysbiosis-associated bacteria, including Prevotella bivia and Gardnerella sp, detected by 16S rRNA sequencing, but not Nugent scoring. Including vaginal pH with the cytokine biomarkers improved the accuracy of the test (82% (95% CI 75% to 88%) correctly classified), although pH alone had poor specificity (61%). CONCLUSIONS: An inexpensive, point-of-care screening test including IL-1α, IL-1ß and IP-10 (and potentially pH) could be used in resource-limited settings to identify women with asymptomatic STIs and dysbiosis. These women could then be referred for aetiological testing, followed by specific treatment.

Clinical validation of the HPVIR high-risk HPV test on cervical samples according to the international guidelines for human papillomavirus DNA test requirements for cervical cancer screening.

BACKGROUND: The indicating FTA card is a dry medium used for collection of cervical samples. HPVIR is a multiplex real-time PCR test that detects 12 high-risk human papillomavirus types (hrHPV) and provides single genotype information for HPV16, - 31, - 35, - 39, - 51, - 56, and - 59 and pooled type information for HPV18/45 and HPV33/52/58. The aim of this study was to evaluate whether a strategy with cervical samples collected on the FTA card and subsequently analysed with the HPVIR test complies with the criteria of the international guidelines for a clinically validated method for cervical screening. METHODS: We performed a non-inferiority test comparing the clinical sensitivity and specificity of the candidate test (FTA card and HPVIR) with a clinically validated reference test (Cobas® HPV test) based on liquid-based cytology (LBC) samples. Two clinical samples (LBC and FTA) were collected from 896 participants in population-based screening. For evaluation of the specificity we used 799 women without ≥ CIN2, and for clinical sensitivity we used 67 women with histologically confirmed ≥ CIN2. The reproducibility was studied by performing inter- and intra-laboratory tests of 558 additional clinical samples. RESULTS: The clinical sensitivity and specificity for samples collected on the FTA card and analysed using the HPVIR test were non-inferior to samples analysed with the Cobas® HPV test based on LBC samples (non-inferiority test score, p = 1.0 × 10- 2 and p = 1.89 × 10- 9, respectively). Adequate agreement of > 87% was seen in both the intra- and inter-laboratory comparisons. CONCLUSIONS: Samples collected on the indicating FTA card and analysed with HPVIR test fulfil the requirements of the international guidelines and can therefore be used in primary cervical cancer screening.

The complete genome sequence of the lumpy skin disease virus vaccine Herbivac LS reveals a mutation in the superoxide dismutase gene homolog.

The lumpy skin disease virus (LSDV) vaccine, Herbivac LS, batch 008, was sequenced and found to differ from the Neethling vaccine strain in the locus encoding a superoxide dismutase (SOD) homolog. The presence of a SOD homolog, be it full-length (as in Herbivac LS) or truncated (as in Neethling) may affect vaccine immunogenicity.

Production of complex viral glycoproteins in plants as vaccine immunogens.

Plant molecular farming offers a cost-effective and scalable approach to the expression of recombinant proteins which has been proposed as an alternative to conventional production platforms for developing countries. In recent years, numerous proofs of concept have established that plants can produce biologically active recombinant proteins and immunologically relevant vaccine antigens that are comparable to those made in conventional expression systems. Driving many of these advances is the remarkable plasticity of the plant proteome which enables extensive engineering of the host cell, as well as the development of improved expression vectors facilitating higher levels of protein production. To date, the only plant-derived viral glycoprotein to be tested in humans is the influenza haemagglutinin which expresses at ~50 mg/kg. However, many other viral glycoproteins that have potential as vaccine immunogens only accumulate at low levels in planta. A critical consideration for the production of many of these proteins in heterologous expression systems is the complexity of post-translational modifications, such as control of folding, glycosylation and disulphide bridging, which is required to reproduce the native glycoprotein structure. In this review, we will address potential shortcomings of plant expression systems and discuss strategies to optimally exploit the technology for the production of immunologically relevant and structurally authentic glycoproteins for use as vaccine immunogens.

Comparative analysis of avian poxvirus genomes, including a novel poxvirus from lesser flamingos (Phoenicopterus minor), highlights the lack of conservation of the central region.

BACKGROUND: Avian poxviruses are important pathogens of both wild and domestic birds. To date, seven isolates from subclades A and B and one from proposed subclade E, have had their genomes completely sequenced. The genomes of these isolates have been shown to exhibit typical poxvirus genome characteristics with conserved central regions and more variable terminal regions. Infection with avian poxviruses (APVs) has been reported in three species of captive flamingo, as well as a free-living, lesser flamingo at Kamfers dam, near Kimberley, South Africa. This study was undertaken to further characterise this virus which may have long term effects on this important and vulnerable, breeding population. RESULTS: Gene content and synteny as well as percentage identities between conserved orthologues was compared between Flamingopox virus (FGPV) and the other sequenced APV genomes. Dotplot comparisons revealed major differences in central regions that have been thought to be conserved. Further analysis revealed five regions of difference, of differing lengths, spread across the central, conserved regions of the various genomes. Although individual gene identities at the nucleotide level did not vary greatly, gene content and synteny between isolates/species at these identified regions were more divergent than expected. CONCLUSION: Basic comparative genomics revealed the expected similarities in genome architecture but an in depth, comparative, analysis showed all avian poxvirus genomes to differ from other poxvirus genomes in fundamental and unexpected ways. The reasons for these large genomic rearrangements in regions of the genome that were thought to be relatively conserved are yet to be elucidated. Sequencing and analysis of further avian poxvirus genomes will help characterise this complex genus of poxviruses.

CCR5 expression, haplotype and immune activation in protection from infection in HIV-exposed uninfected individuals in HIV-serodiscordant relationships.

Several host factors have been implicated in resistance to HIV infection in individuals who remain HIV-seronegative despite exposure. In a cohort of HIV-serodiscordant heterosexual couples, we investigated interactions between systemic inflammation and T-cell activation in resistance to HIV infection. Males and females in stable long-term relationships with either HIV-infected or uninfected partners were recruited, blood T-cell activation (CD38, HLA-DR, CCR5 and Ki67) and plasma cytokine concentrations were evaluated. The HIV-negative exposed individuals had significantly lower frequencies of CCR5+ CD4+ and CD8+ T cells than unexposed individuals. Mean fluorescence intensity of CCR5 expression on CD4+ T cells was significantly lower in HIV-negative exposed than unexposed individuals. Protective CCR5 haplotypes (HHA/HHF*2, HHF*2/HHF*2, HHC/HHF*2, HHA/HHA, HHA/HHC and HHA/HHD) tended to be over-represented in exposed compared with unexposed individuals (38% versus 28%, P = 0·58) whereas deleterious genotypes (HHC/HHD, HHC/HHE, HHD/HHE, HHD/HHD and HHE/HHE) were under-represented (26% versus 44%; P = 0·16). Plasma concentrations of interleukin-2 (P = 0·02), interferon-γ (P = 0·05) and granulocyte-macrophage colony-stimulating factor (P = 0·006) were lower in exposed compared with unexposed individuals. Activation marker expression and systemic cytokine concentrations were not influenced by gender. We conclude that the dominant signature of resistance to HIV infection in this cohort of exposed but uninfected individuals was lower T-cell CCR5 expression and plasma cytokine concentrations.

Xenogenic rolling-circle replication of a synthetic beak and feather disease virus genomic clone in 293TT mammalian cells and Nicotiana benthamiana.

The preparation of infectious beak and feather disease circovirus virions (BFDV) has until now relied on the extraction of virus from whole tissue of deceased or euthanized parrots known to be infected with the virus. Extraction from diseased tissue is necessary, as the virus has yet to be grown in vitro using tissue-cultured cells from any source. While infectious DNA clones have been synthesized for porcine and duck circoviruses, and both replicate in host cells and result in active viral infection in animals, this has not been shown for BFDV. The aim of this study was to prepare an infectious BFDV genomic clone that could be used as challenge material in birds for vaccine testing. A putatively infectious BFDV genomic clone was designed and tested in mammalian cell culture, and in the plant Nicotiana benthamiana in the presence of plant-specific ssDNA geminivirus replication components. Replication was assessed using rolling-circle amplification, qPCR, replication-deficient clones and rescue plasmids. We showed that a synthetic partially dimeric BFDV genomic clone self-replicated when transfected into 293TT mammalian cells, and was also replicated in N. benthamiana in the presence of geminivirus replication elements. This is the first report of a BFDV genome replicating in any cell system, and the first report of a circovirus replicating with the aid of a geminivirus in a plant. Both of these developments could open up possibilities for making reagents and vaccines for BFDV, testing vaccine efficacy and investigating viral replication using rationally designed artificial genomes.

Six host-range restricted poxviruses from three genera induce distinct gene expression profiles in an in vivo mouse model.

BACKGROUND: Host-range restricted poxviruses make promising vaccine vectors due to their safety profile and immunogenicity. An understanding of the host innate immune responses produced by different poxvirus vectors would aid in the assessment, selection and rational design of improved vaccines for human and veterinary applications. Novel avipoxviruses are being assessed to determine if they are different from other poxvirus vectors. Analysis of the transcriptome induced in a mouse model would aid in determining if there were significant differences between different poxvirus vectors which may reflect different adjuvant potential as well as establish if they should be further evaluated as vaccine vectors. RESULTS: We compared host transcript abundance in the spleens of BALB/c mice twenty four hours after intravenous infection (10(5) pfu/mouse) with six host-restricted poxvirus species from three genera, namely Lumpy Skin Disease virus (LSDV), Canarypox virus (CNPV), Fowlpox virus (FWPV), modified vaccinia Ankara (MVA) and two novel South African avipoxviruses, Feral Pigeonpox virus (FeP2) and Penguinpox virus (PEPV). These six viruses produced qualitatively and quantitatively distinct host responses with LSDV, followed by MVA, inducing the greatest interferon (IFN) response. FeP2 and PEPV caused very little change to host transcript abundance compared to the other 4 viruses tested. CNPV and FWPV induced the up regulation of two immunoglobulin genes (Ighg and Ighg3 (IgG3)) with CNPV inducing a third, Ighm (IgM). HIV-1-specific IgG3 antibodies have been correlated with decreased risk of HIV-1 infection in the RV144 trial, which included a CNPV-based vector (Yates et al. (Sci Transl Med, 6(228) p228, 2014). Up regulation of IgG3 by CNPV and FWPV but not the other poxviruses tested in vivo, implies that these two avipoxvirus-vector backbones may be involved in stimulation of the clinically important IgG3 antibody subclass. Differential transcript abundance associated with the different poxviruses is further discussed with particular emphasis on responses related to immune responses. CONCLUSION: Six, genetically diverse host-restricted poxviruses produce different responses in a mouse model early after infection. These differences may affect the immune response induced to vaccine antigen in vectors based on these viruses. The two novel avipoxviruses were clearly distinguishable from the other viruses.