Archive for the ‘HIV’ Category

Legends of Virology

31 January, 2014

I have been fortunate enough this week to be in Pretoria, at the first Animal and Human Vaccine Development in South Africa Conference (Twitter #AHVDSA): partly because it is a very timeous and necessary meeting to help to establish strategies for this purpose, and partly because there is a significant presence of some legendary figures of international and South African virology.

Marc van Regenmortel – who we count as local even if he lives in Strasbourg – helped Bob Millar and others at the University of Pretoria to organise this meeting. He also used the opportunity of having a bunch of old virological friends visiting him at the University of Stellenbosch’s STIAS to bolster the conference presentations.

So it was that we have Errling Norrby of Sweden with us; we have Fred Murphy of Ebola fame; Marian Horzinek of veterinary virology repute; Marc himself, our iconoclastic viral immunologist; Jose Esparza of the BMG and an eminent poxvirologist – and Jean-Marie Andrieu, an oncologist with an interest in tolerogenic HIV vaccines.

Local legends are present too: we have Daan Verwoerd, legendary orbivirologist and former Director of the venerable and distinguished Onderstepoort Veterinary Institute; Henk Huismans, who did the first molecular work on orbiviruses in the 1970s, and is still active; Bob Swanepoel, doyen of the African haemorrhagic fever viruses.

Good people.

Oh, and of course, me and Anna-Lise Williamson; Dion du Plessis of OVI; Lynn Morris of the NICD; Albie van Dijk of UNW; Glenda Gray of the MRC, among 150 delegates

A great meeting, all in all, and very timely, given the contents of the SA Governmental Bioeconomy Strategy document released recently.

20140131-120134.jpg

Legends alive: from left, Fred Murphy; Daan Verwoerd; Bob Millar; Henk Huismans; Errling Norrby; Marc van Regenmortel
20140131-120151.jpg

Jean-Marie Andrieu; Marc van Regenmortel – at a VERY good unofficial dinner

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Legends and friends at supper: Marc, Fred, Eric Etter (CIRAD); Jose Esparza; Marian Horzinek; Errling, Anna-Lise Williamson

14 adults ‘cured’ of killer HIV virus [NOT!!]

16 March, 2013

See on Scoop.itVirology News

TWO weeks after doctors rid a baby of the disease, it appears the treatment has worked on full-grown men and women

Ed Rybicki‘s insight:

You have to hate sub-editors – the people who are tasked, in papers like the Sun, to come up with the most lurid headline possible.

 

The facts are these: a number of people were treated, soon after infection with HIV-1, with a course of combo ARVs.  For one reason or another, they stopped taking them – and they are, up to seven years out – controlling their virus load to undetectable levels.

 

Note: they are almost certainly NOT cured; the virus is integrated into their CD4+ T-cells, and is simply quiescent or ticking over at a very low level of expression.

 

Howevr, it is potentially good news – IF it can be replicated in a wider cohort, and IF people can be caught at an early stage of infection.

See on www.thesun.co.uk

ViroBlogy: 2012 in review

1 February, 2013

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

Click here to see the complete report.

Scientists Find Compound That Helps HIV, Flu Vaccines – Health News – redOrbit

28 August, 2012

See on Scoop.itVirology 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

PLoS Pathogens: ADCC Develops Over Time during Persistent Infection with Live-Attenuated SIV and Is Associated with Complete Protection against SIVmac251 Challenge

24 August, 2012

See on Scoop.itVirology 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

Vaccination with Adenovirus Serotypes 35, 26, and 48 Elicits Higher Levels of Innate Cytokine Responses than Adenovirus Serotype 5 in Rhesus Monkeys

24 August, 2012

See on Scoop.itVirology 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

Gag-Specific Cellular Immunity Determines In Vitro Viral Inhibition and In Vivo Virologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys

24 August, 2012

See on Scoop.itVirology 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

What’s Causing the Spike in HIV Infection in Old Chinese Men? – Business Insider

23 August, 2012

See on Scoop.itVirology News

China DailyWhat’s Causing the Spike in HIV Infection in Old Chinese Men?

See on www.businessinsider.com

Radical Therapy for HIV-Infected People?

18 May, 2012

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.

The origin of HIV: still so much garbage out there

20 February, 2012

While curating Virology News today, I came across another reprocessing of new that I had come across earlier concerning apparent natural protection of some African female sex workers against HIV infection: this was the intriguingly-entitled “African women’s genitals provide clue to HIV prevention“, in what appears to be an online Nigerian newspaper.

This recapitulates, very accurately, the information I reported in Virology News, which was the subject of a news release following the publication in the September 2011 edition of PLoS One of a study entitled “High Level of Soluble HLA-G in the Female Genital Tract of Beninese Commercial Sex Workers Is Associated with HIV-1 Infection”.  The gist of this is that:

“HIV-resistant sex workers in Africa have a weak inflammatory response in their vaginas – a surprise for the researchers, who were expecting the contrary considering the women’s high exposure to the virus.”

This may lend further credence to the observation that progression to AIDS in HIV-infected people is associated with a state of chronic immune activation – and that SIV-infected vervet monkeys do not exhibit such chromic immune activation, and do not progress like humans do.

What is interesting about the Nigerian article, however, is not what it reports – it is the online comments that follow it.  Here is a selection:

“Was HIV realy discovered in Africa ? Forget the western media propaganda . I have believed , for years , that HIV is a laboratory virus designed for genocide in the thick of apartheid inhuman policies in South Africa .”

“Neither did HIV originate  nor was it perculiar to Africa. It was the creation of the Western countries to stsyematically reduce African population. that the subjects of this study were exposed to HIV virus attests to this fact.”

And my personal favourite:

“So you have already swallowed up the white propaganda that the AIDS virus was first discovered in 1981 in a remote area of central Africa in the green monkey!  A fairy tale, which never explains why prior to its first clinical detection among western homosexual men in the late seventies, no case was found in Africans, and no animal or human population died off in Africa, yet the homosexual population of the west was seriously threatened until their protected sex campaign took off.

You must be unaware that about 35 years ago the Soviet KGB told the world the truth about AIDS….

Jakob Segal, a former biology professor at Humboldt University in then-East Germany, proposed that HIV was engineered at a U.S. military laboratory at Fort Detrick, by splicing together two other viruses, Visna and HTLV-1. According to his theory, the new virus, created between 1977 and 1978, was tested on prison inmates who had volunteered for the experiment in exchange for early release. He further suggested that it was through these prisoners, most of who were homosexuals, that the virus was spread to the population at large.”

What is depressing is that there is just one comment saying “…where HIV started is of little significance now. the issue is that our brothers Africans are the ones affected so we must work hard to find the cure and save our brothers.”

What is obvious is that, even in an environment such as one of the most developed nations in Africa, where intelligent science reporting is happening, the public seems to be alarmingly misinformed about the origin of HIV and predisposed to believe racist conspiracy theories that were debunked years ago.

FACT:
HIV did not come from “green monkeys” and was not discovered in 1981: the virus was described in 1983 and 1984, and HIV entered the  human population in central Africa multiple times, from chimpanzees and possibly also from gorillas, almost certainly via bushmeat – and this happened in the 1930s or even earlier.

FACT:
HIV could not possibly have resulted  from the splicing together of Visna virus and HTLV-1, as no HIV sequence bears any strong resemblance to either virus – and especially not to both of them in different parts of their genomes, as they would be expected to if they were artificial recombinants.  Moreover, the first HIV that has been reliably dated comes from a sample taken in the Congo in 1959.

All of these facts can be easily discovered by a trawl of either the scientific literature, or a first-level digest of that literature by reputable journalists.  All else is fiction…and malicious fiction at that, whether or not such supposed luminaries as Thabo Mbeki believe it.


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