So: thank you, anyone who clicked in, and regular visitors. You make it worthwhile!!
The WordPress.com stats helper monkeys prepared a 2012 annual report for this blog.
Here’s an excerpt:
4,329 films were submitted to the 2012 Cannes Film Festival. This blog had 33,000 views in 2012. If each view were a film, this blog would power 8 Film Festivals
Introduction:
Haemagglutinin-only Influenza A virus VLP. Courtesy of Russell Kightley Media
The new international conference on virus-like particles and nano-particles (VLPNPV) took place in Cannes, France at The Novotel Montfleury Hotel from the 28th to the 30th of November 2012. The scope of the conference included virus-like particles (VLPs), the plant-based expression of VLP vaccines as well as expression and optimisation of VLPs.
Other topics included in the conference were:
A multitude of topics were covered during the conference and many of the talks pertained to the immunogenicity of the VLPs and nano-particles and how they compared with the immunogenicity of DNA or subunit vaccines.
Talks were given by researchers from companies such as Medicago, Mucosis, Pevion Vaccines and Novavax. These talks gave a perspective on factors that need to be considered when commercialising VLP/nano-particle vaccines and to be GMP compliant.
Compelling presentations:
Developing plant-made virus-like particle vaccine products: An integrated platform from discovery to commercial scale
Marc-Andre D’Aoust, Nathalie Landry, Sonia Trepanier, Michele Dargis, Manon Couture and Louis-Philippe Vezina (Medicago, Quebec City, Quebec, Canada)
This talk was about a plant-made VLP against both pandemic and seasonal influenza- these vaccines are now in the clinical trial phase. What was especially interesting was the view from an industry point of view where expression had to be scaled up to produce large amounts of vaccine. The Medicago platform can synthesize and clone approximately 100 gene constructs in two weeks, they can prepare 100 bacterial cultures per week and they have automated infiltration where 200 plant transformations can be performed per day and 150 VLP engineering approaches can be tested in one week. For influenza Medicago tested 48 different infiltration approaches in one day for HA, NA, M1, M2 as well as P1 Gag and HGalT. Medicago has been able to produce 10 million doses of HA VLPs in just one month.
See also:
Development of RNA-free plant VLPs a source of novel therapeutics
George Lomonossoff (John Innes Centre, Norwich, UK)
This group made empty Cowpea Mosaic Virus (CPMV) VLPs that contained no RNA. CPMV VLPs are versatile nanoparticles to which organic, inorganic and biological molecules can be bound. The empty nature of the particle means that they can be used as carrier molecules for therapies; this could prove to be potentially useful as a cancer-treatment therapy. The system is advantageous because of the lack of RNA which makes the particles non-infectious and no bio-containment is needed for the production of these VLPs.
Immunogenicity of VLPs: an immunological perspective
Martin Bachmann (University of Zurich, Zurich, Switzerland)
Background was given from immunological point of view about what makes VLPs so immunogenic. Three properties contribute to the immunological properties of VLPs (1) their size, (2) the repetitiveness of the particle capsid which provides multiple sites for antibody binding and (3) TLR ligands – the particle can be disassembled, the RNA removed and replaced with a TLR ligand to enhance immunogenicity. Also, the size of VLPs is optimal for drainage to the lymph nodes.
Immunogenicity optimization strategies for public-sector development of vaccines: the critical role of optimizing the antigen.
Martin Howell Friede (WHO, Geneva, Switzerland)
This talk was about looking at VLPs from the vaccine development view. Monomeric antigens are not very immunogenic; therefore adjuvants were developed and came into use. For an efficient vaccine the antigen must be multimeric as antigen alone is insufficient to be immunogenic without adjuvant. Two factors have to be considered when producing a vaccine for FDA approval; (1) optimise the antigen before using an adjuvant, (2) use an adjuvant that has already been approved by the FDA. VLPs as vaccines provide the potential for immune-stimulation without the addition of adjuvant as the multimeric presentation of the antigen will enhance its immunogenicity.
Enhancing the immunogenicity of VLP vaccines
Richard W. Compans (Emory University, Atlanta, Georgia, USA)
This talk highlighted strategies which could be used to enhance the immunogenicity of VLPs.
See also:
Innate and adaptive responses to plant-made VLP vaccines
Brian Ward (McGill University, Montreal, Quebec, Canada)
Brain Ward is also the medical officer at Medicago. Humans rarely react to plant proteins/antigens. The plant glycans fucose/xylose at the N-terminal is an allergen and can cause anaphylaxis in humans. During trial experiments with influenza no individuals developed IgE responses to plant glycans, therefore plant produced vaccine is safe. The H1 VLP induced long lasting memory multifunctional T-cell responses in humans.
Impressions of the conference:
The conference was well organised with leaders in the field presenting their work. Interaction with the delegates aid in building crucial networking opportunities and work relationships. The international arena is packed with new technology development allowing us the opportunity to learn and improve our own understanding of various concepts.
This conference proved to be an invaluable learning experience and I thank the NRF for this opportunity and for providing me with the funding to attend this conference. The exposure to conferences, especially those in the international arena, aid in the development of students and contribute to the quality of research that is conducted at UCT.
References:
1. VLPNPV website
(http://www.meetingsmanagement.co.uk/index.php?option=com_content&view=article&id=33&Itemid=83)
2. Personal notes taken at the conference
See on Scoop.it – Virology News
“Oxford University scientists have discovered a compound that greatly boosts the effect of vaccines against viruses like flu, HIV and herpes in mice.”
Well, no, theyt haven’t: what they HAVE done is find that a very well known chemical has activity as an adjuvant – and very strong activity, it appears.
“The Oxford University team found that PEI, a standard polymer often used in genetic and cell biology, has strong adjuvant activity.
redOrbit (http://s.tt/1lPjE)”
It is also useful as a mucosal adjuvant, which is very useful for intranasal / oral vaccination strategies.
See on www.redorbit.com
See on Scoop.it – Virology and Bioinformatics from Virology.ca
“Here we show that live-attenuated SIV induces progressive increases in ADCC over time, and that the development of these antibodies is dependent upon the persistent replication of the vaccine strain. In two different experiments, the animals immunized with live-attenuated SIV that remained uninfected after pathogenic SIV challenge had higher measures of ADCC than those that became infected. Our results suggest that antibodies contribute to protection by live-attenuated SIV, and that persistent stimulation of antibody responses may be essential for HIV-1 vaccines to induce high ADCC activity.”
Shit HOT results, in that they demonstrate that – as some have said repeated ly over years – that neutralising Ab are NOT necessarily the Holy Grail, and that ADCC and other mechanisms are also really important. Good Stuff…B-)
See on www.plospathogens.org
See on Scoop.it – Virology and Bioinformatics from Virology.ca
“These data demonstrate that Ad35, Ad26, and Ad48, which utilize CD46 as their primary cellular receptor, induce significantly greater innate cytokine responses than Ad5, which uses the coxsackievirus and adenovirus receptor (CAR). These differences in innate triggering result in markedly different immunologic milieus for the subsequent generation of adaptive immune responses by these vaccine vectors.”
Important news for the vectored vaccine community in general, and for HIV vaccine in particular: Ad5 was the vehicle of choice; now it looks as though it shouldn’t be.
Adenovirus graphic courtesy of Russell Kightley Media
See on jvi.asm.org
See on Scoop.it – Virology News
“We observed that CD8+ lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV in vitro. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4+ and CD8+ T lymphocyte responses. These findings demonstrate that in vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in vivo virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.”
In other words: having Gag or a gag gene included in a vaccine against SIV given to monkeys was more important than having Pol or Env when it came to control of virus replication – although, as has been shown elsewhere, Env responses are important for protecting against acquisition. This has important implications for human vaccines – although “monkeys aren’t men, and mice lie” – and in particular for the South African SAAVI vaccines, which elicit quite good Gag-specific cellular responses.
We wait in hope. Graphic showing immune cells associated with HIV control courtesy of Russell Kightley Media.
See on jvi.asm.org
See on Scoop.it – Virology News
China DailyWhat’s Causing the Spike in HIV Infection in Old Chinese Men?
See on www.businessinsider.com
I started working on human papillomaviruses (HPVs) some 22 years ago, back at the dawn of PCR: I helped my then-new major collaborator (and wife of 2 years), Anna-Lise Williamson, design some degenerate primers for amplifying as wide a range as possible of high-risk HPVs from cervical biopsy samples. These worked pretty well, and are still highly useful for the purpose, despite the many novel types found since then.
We went on to do another two papers together on looking at variation and typing of HPVs via PCR and and sequencing, then took a deviation into making candidate vaccines for HPV and HIV. Anna-Lise carried on with surveilling for HPVs, however, and has ended up with a WHO Regional Laboratory for HPV work. She also started working on HPV infections in HIV-infected women: work on a study cohort showed that while HIV-free women usually had only 1 HPV type, the 109 HIV-infected often were infected with multiple HPV types. In association with Anna Salimo in my lab, we started a deep sequencing pilot study on the sample with the most HPVs. This turned this into a regional study, with help on assembling and interpreting sequence data from Prof Johan Burger’s lab at the University of Stellenbosch, and it was revelatory: while a commercial kit could detect 12 HPV types in one sample, next-gen sequencing found 16.
We went on to do PCR on all 109 samples in the cohort with specific primers for the types not found by the kit, and showed prevalences up to 15% in the HIV-infected group. This is an important result, because otherwise-innocuous HPV types that do not show up in normal women, may well be associated with disease in the HIV-infected – and will probably not be protected against by the current HPV vaccines.
We continue to do work on these samples, and it will be very interesting to see what the new methodologies show up. Especially as sequencing becomes cheaper, and we can do more samples…! Meantime, we have published the pilot study:
Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women.
Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR.
Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence’s of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6 %, 12.8 %, 4.6 % and 8.3 % respectively.
Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.
I thank Russell Kightley Media for use of the HPV and cervical cancer graphic.
See on Scoop.it – Virology News
Eighty per cent of the meat eaten in Cameroon is killed in the wild and is known as “bushmeat”. The nation’s favoured dishes are gorilla, chimpanzee or monkey because of their succulent and tender flesh. According to one estimate, up to 3,000 gorillas are slaughtered in southern Cameroon every year to supply an illicit but pervasive commercial demand for ape meat .
“Everyone is eating it,” said one game warden. “If they have money they will buy gorilla or chimp to eat.”
Frankie, a poacher in the southern Dja Wildlife reserve who gave a fake name, said he is involved in the trade because he can earn good money from it, charging around £60 per adult gorilla killed. “I have to make a living,” he said. “Women come from the market and order a gorilla or a chimp and I go and kill them.”
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This is a big deal – and not because as, in the words of the article, “Britain is at risk from an outbreak caused by the lethal Ebola or Marburg viruses contained in illegal imports of bush meat from Africa”.
Because AFRICA is at risk from such diseases – and the death toll will be much higher here, where the standard of care is so much lower than in Europe.
And because we are losing the closest relatives that we as humans have – to human greed for meat. How despicable, and how sad, is that?
See on www.independent.co.uk
To mark HIV Vaccine Awareness Day, 18th May – Journal Club – Lucian Duvenage:
Excision of HIV-1 Proviral DNA by Recombinant Cell Permeable Tre-Recombinase
Mariyanna, L., Priyadarshini, P., Hofmann-Sieber, H., Krepstakies, M., Walz, N., Grundhoff, A., Buchholz, F., Hildt, E., Hauber, J., 2012. Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase. PloS One 7, e31576.
Introduction
HIV Life Cycle. Russell Kightley Media, http://www.rkm.com.au
Highly active antiretroviral therapy (HAART) is a combination of drugs that has significantly elongated the lifespan HIV-infected people. HAART targets viral reverse transcriptase, protease and integrase. There are disadvantages including drug toxicity and the appearance of drug resistant HIV strains in people not adhering to or withdrawing from their treatment. There is a need for new therapies that not only block virus replication but also eliminate HIV from persistent viral reservoirs. An attractive option is Tre-recombinase, which been shown to excise provirus from the genomic DNA of infected cell cultures. The development of Tre recombinase is a previous publication (Sarkar, I., Hauber, I., Hauber, J., Buchholz, F., 2007. HIV-1 proviral DNA excision using an evolved recombinase. Science 316, 1912-5.)
The Tre recombinase was created from the Cre recombinase which is a well-known tool in mouse genetics. The authors were able to alter the specificity of the enzyme by many cycles of directed protein evolution.
The Cre recombinase precursor removes genomic DNA that is flanked by two loxP sites by recombination. The authors were able to alter the specificity for loxP sites to HIV-1 LTR (long terminal repeat) sites. LoxP and the HIV LTR had 50% sequence similarity.
The main problem with the development of antiviral agents is the delivery to infected cells in vivo, without causing adverse side effects. There are many reported technologies for the delivery of macromolecules such as proteins, nucleic acids or peptides. The most popular currently is the use of protein transduction domains (PTD) also known as cell penetrating peptides (CPP) from different sources. These have been useful for the delivery of various genes and proteins, including site-specific recombinases. The best studied and most applied PTD’s are peptides derived from the basic domain of HIV-1 Tat. But recently a powerful cell permeable translocation motif (TLM) has been described. This TLM is derived from a hepatitis B virus surface antigen. This TLM peptide is able to enter cells very efficiently, without affecting the integrity of the cells, or interfering with intracellular signal transduction cascades.
This paper describes the delivery of Tre-recombinase into cells using these PTD’s including HIV Tat and the HBV TLM. These so-called cell-permeable Tre-recombinases could eventually be useful for antiretroviral therapy, especially for virus eradication.
Results
Proteins
Different protein fusions were created and expressed in E. coli ; Tre-recombinase is fused to His tag, with/without nuclear localisation signal (NLS) and with the PTD (HIV Tat) or TLM (translocation motif derived from hepatitis B) or TLM as an inverted repeat.
They tested the cellular toxicity of the protein at their highest concentration by alarmBlue assay in HeLa cells. The proteins were incubated with the cells for 48 h. None of the proteins had any significant effect on the cellular metabolism
Cell permeability
Interestingly, all of the proteins entered cells, even those without a PTD or nuclear translocation signal. The authors explain that the Cre enzyme precursor to Tre has been shown to transduce into mammalian cells without any help, and therefore it is likely that the Tre enzyme shares this property. The authors did remark that the signal intensities were higher for those proteins with a PTD, indicating higher transduction efficiency.
Analysis of Tre activity in HeLa cells
A transient reporter assay demonstrated the activity of the Tre fusions: The reporter construct contains the target LTR sites that flank a puromycin resistance gene. Tre enzyme activity results in the loss of this gene, and gives a smaller PCR product using primers that anneal to the vector backbone. Cells transfected with the reporter construct were incubated with the 1 µM of the various proteins for 5 hours. The positive control was co-transfected with a construct expressing the Tre enzyme. PCR was performed on DNA extracted from cells after 48 hours. The presence of the smaller PCR product indicated that recombination had happened, as in the positive control. All of the proteins had varying degrees of activity, but notably the protein with the TLM PTD had the highest activity, with no un-recombined product detected by the PCR.
The authors went on to demonstrate that the Tre fusion proteins were active on at the genomic level, i.e. on chromosomal DNA. They used cells with the reporter construct was stably integrated into the genome.
Interaction of proteins with LTR sequences in living T-cells
Co-immunoprecipitation (ChIP) assays were done on using HIV-1 -infected T-cells (CEM-SS) to demonstrate the interaction of two of the Tre fusion proteins with the HIV LTR target sequences. The results showed that the proteins interacted with target LTR sequences in the genome of infected T-cells.
Microarray
The authors performed a transcriptome anaylsis on cells exposed to the Tre fusion proteins, using human whole genome microarrays. They concluded that the proteins were unlikely to have a significant effect on gene expression in the host cells, as very few genes were regulated more than 2.5-fold.
Recombination of the full-length HIV proviral genome:
Up to this point, the Tre fusion proteins had been shown to be capable of excising reporter construct gene flanked by LTR sequences both at the episomal level and the chromosomal level. The authors also showed that the proteins bind to the target sequences in HIV-infected living T cells.
It was essential that the Tre fusions could excise that HIV proviral genome from the chromosomal DNA of HIV-infected cells. The aouthors generated HeLa cells and T cells infected with pseudotype HIV-1. These are cells with the full-length HIV provirus integrated into the genome.They chose one of their proteins (TLM fusion showing highest activity in the reporter assays) for transduction into these cells. After transduction, PCR was performed to detect the HIV circular recombination product. They found that the recombination activity increased in a dose-dependent manner in both the HeLA cells and the T-cells. They also sequenced the PC products and were able to confirm HIV sequences.
Discussion
Some novel therapies for the treatment of HIV focus on the eradication of the virus in infected individuals. These include RNA-based technologies such as RNA aptamers, siRNA and ribozymes, but while these have shown to reduce viral load and viral replication, they have so far failed in virus eradication. A recent approach aimed at virus eradication is the reduction of surface CCR-5 receptors, through the expression of engineered zinc finger nucleases. This results in fewer CCR-5 surface receptors and could prevent new infection by CCR-5 tropic HIV.
The other approach is the use of site specific recombinases like Tre, which can excise the provirus from the host genome, thus potentially eradicating the virus from the individual. Ideally, the gene expressing Tre could be delivered to and expressed in target cells using a viral vector. But there are safety concerns as most of these are derived from pathogenic viruses. Therefore it may be advantageous to deliver the Tre enzyme directly to host cells. One way of doing this is through protein translocation domains (PTD’s). Protein transduction domains (PTD’s) can deliver bioactive molecules, including genes, siRNA, proteins or liposomes into all types of cells in vitro and furthermore into various organs in vivo. But they have not been applied yet for human use. PTD’s are easily fused to any target protein through cloning and expression of the fusion protein. The LTM used in this paper, derived from hepatitis B virus surface antigen, has low immunogenicity and high spreading capacity.
One strategy of using cell-permeable Tre enyme could be to harvest T-cells from the patient by apheresis and transduce them in vitro. They could then potentially be expanded and reinfused into the patient. This could complement or even replace gene transfer procedures.
In this paper the expressed Tre fusion proteins could enter cells and act on the target sequences to excise the HIV provirus from the genome, but the Tat fusion and the TLM fusion had higher activity than those that didn’t have a PTD tag. In particular, fusion to the newly described domain (TLM) from hepatitis B was resulted highest activity. This may explained by the fact that, in contrast to HIV Tat, TLM does not rely on endocytosis to enter cells. This might preserve enzyme activity and protein half-life.
In conclusion, cell permeable Tre enzyme could eventually be useful as an anti-HIV therapy in the post-HAART era.
I have been at UCT since I came to Cape Town on holiday from Zambia in 1974, and fell in love with the place. I obtained a BSc in Chemistry and Microbiology in 1976, an Hons in Virology in 1977, an MSc in Virology in 1979, and a PhD in the same discipline in 1984. I became a Lecturer in Virology in 1981, and have risen through the ranks to become a Professor in Microbiology (in January 2003). I am also a Founder Member of the Institute of Infectious Disease and Molecular Medicine (IIDMM) based in the Health Sciences Faculty.
