Posts Tagged ‘HIV’

African monkey meat that could be behind the next HIV

25 May, 2012

See on Scoop.itVirology 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

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.

I repeat: HIV causes AIDS

14 March, 2012

My participation in an acrimonious exchange in the Comments section of a recent article in The Scientist by UCT’s own Nicoli Nattrass has brought something home to me that I had hoped was long dead.  That is, that there are some apparently quite well-educated people who still believe that HIV either does not actually exist, or that it does not cause Acquired Immune Deficiency Syndrome (AIDS).

I find this almost unbelievable: that people in this day and age can make statements like

  • “HIV has never been purified and isolated properly, the tests accordingly have no virological gold standard, but are validated against each other in a wholly circular fashion”, and
  • “After 25 years HIV experts have yet to come up with an agreed method of action (how HIV causes AIDS)”, and
  • “Even if I were to accept that there is such a thing as a unique, coherent viral entity HIV that infects T-cells, it proves nothing”,

boggles my mind.  There are  236 870 papers at least in the scientific literature since the early 1980s on HIV: are we to understand that probably over a million authors from al over the world have willingly participated in a fraud that has to be the biggest ever perpetrated?  Or do we believe that there is something called HIV, that it causes AIDS, and that the overwhelming proportion of the world’s scientists have no problem with this?

I know what I think – and I am going to reprise it here.  I put this together in 2001 or thereabouts; however, nothing has changed.  HIV still exists, it still causes AIDS, and anyone who denies this is truly, truly invincibly ignorant.

Human Immunodeficiency Virus (HIV) is a typical retrovirus of the genus Lentivirus, family Retroviridae: these are viruses which have single-stranded RNA as their genetic material, but multiply via a double-stranded DNA intermediate.  The replication and classification of such viruses is discussed in greater detail on this site, as well as at the Leicester site.

The virus infects its target cells – which happen to mainly be helper T-lymphocytes, because these are the cells which have the highest density of the preferred receptor molecule – by specifically attaching to a protein carried on the surface of the cells known as CD4.  This is shown hereAn animated view of the whole infection process is shown here; a more detailed schematic animated view here.  The process is discussed in detail here; however, what happens is that virus particles fuse with the target cells, and deliver their genetic material plus inside.  This RNA is then converted into DNA by means of a unique viral enzyme called reverse transcriptase.  The double-stranded DNA is then inserted randomly into a host cell  chromosome by means of another viral enzyme (integrase).  As part of the host chromosome it is expressed exactly the same way as host DNA – AND IS NOW PART OF THE CELL AND CANNOT BE REMOVED.  This means that infected cells are infected for life, which is different to most other types of virus infections: cells harbouring viral DNA continue to make infectious virus particles until they die or are killed.

SEE HERE FOR GRAPHICAL REPRESENTATION OF THE CELLULAR INFECTION PROCESS (Courtesy Russell Kightley)


Definitions of AIDS


From the National Institute of Allergy and Infectious Diseases (NIAID) site

niaid.gif (8285 bytes)

“An HIV-infected person is diagnosed with AIDS when his or her immune system is seriously compromised and manifestations of HIV infection are severe

“The U.S. Centers for Disease Control and Prevention (CDC) currently defines AIDS in an adult or adolescent age 13 years or older as the presence of one of 26 conditions indicative of severe immunosuppression associated with HIV infection, such as Pneumocystis carinii pneumonia (PCP), a condition extraordinarily rare in people without HIV infection. Most other AIDS-defining conditions are also “opportunistic infections” which rarely cause harm in healthy individuals.

A diagnosis of AIDS also is given to HIV-infected individuals when their CD4+ T-cell count falls below 200 cells/cubic millimeter (mm3) of blood

Healthy adults usually have CD4+ T-cell counts of 600-1,500/mm3 of blood. In HIV-infected children younger than 13 years, the CDC definition of AIDS is similar to that in adolescents and adults, except for the addition of certain infections commonly seen in pediatric patients with HIV. (CDC. MMWR 1992;41(RR-17):1; CDC. MMWR 1994;43(RR-12):1).

In many developing countries, where diagnostic facilities may be minimal, healthcare workers use a World Health Organization (WHO) AIDS case definition based on the presence of clinical signs associated with immune deficiency and the exclusion of other known causes of immunosuppression, such as cancer or malnutrition

An expanded WHO AIDS case definition, with a broader spectrum of clinical manifestations of HIV infection, is employed in settings where HIV antibody tests are available (WHO. Wkly Epidemiol Rec. 1994;69:273).”


Evidence That HIV Causes AIDS


There is very little doubt among the vast majority of people working with HIV or with people with AIDS, that HIV causes AIDS (see above).  The media tended to present everything in terms of a two-sided debate, with equal credence being given to both “sides”.  This is very misleading for the population at large, as they are presented with two sets of information that receive equal billing – and they are not qualified to judge between the viewpoints.

Quite simply, there is a vast body of evidence supporting the proposition that HIV causes AIDS

I have presented some of the most compelling below; there is an enormous body of other evidence available on the Web, let alone in medical libraries.  The same cannot be said for the alternative view(s).  Perhaps the “best” of the denialist sites is is probably the “Rethinking AIDS” site.   This has a wealth of (dis)information on the subject, including such gems as the following, from Kary Mullis:

“If there is evidence that HIV causes AIDS, there should be scientific documents which either singly or collectively demonstrate that fact, at least with a high probability. There is no such document.” (Sunday Times London, 28 nov. 1993).

That this statement is nonsense is amply demonstrated by the following:

From the Durban Declaration site:

“The evidence that AIDS is caused by HIV-1 or HIV-2 is clear-cut, exhaustive and unambiguous.  This evidence meets the highest standards of science.  The data fulfill exactly the same criteria as for other viral diseases, such as poliomyelitis, measles and smallpox:

  • Patients with acquired immune deficiency syndrome, regardless of where they live, are infected with HIV.
  • If not treated, most people with HIV infection show signs of AIDS within 5-10 years.  HIV infection is identified in blood by detecting antibodies, gene sequences or viral isolation.  These tests are as reliable as any used for detecting other virus infections.
  • Persons who received HIV-contaminated blood or blood products develop AIDS, whereas those who received untainted or screened blood do not.
  • Most children who develop AIDS are born to HIV-infected mothers.  The higher the viral load in the mother the greater the risk of the child becoming infected.
  • In the laboratory HIV infects the exact type of white blood cell (CD4 lymphocytes) that becomes depleted in persons with AIDS.
  • Drugs that block HIV replication in the test tube also reduce viral load and delay progression to AIDS.  Where available, treatment has reduced AIDS mortality by more than 80%.
  • Monkeys inoculated with cloned SIV DNA become infected and develop AIDS.

Further compelling data are available.  HIV causes AIDS.  It is unfortunate that a few vocal people continue to deny the evidence.  This position will cost countless lives.”

From the NIAID site (excerpted):

AIDS and HIV infection are invariably linked in time, place and population group.

Historically, the occurence of AIDS in human populations around the world has closely followed the appearance of HIV. In the United States, the first cases of AIDS were reported in 1981 among homosexual men in New York and California, and retrospective examination of frozen blood samples from a U.S. cohort of gay men showed the presence of HIV antibodies as early as 1978, but not before then. Subsequently, in every region, country and city where AIDS has appeared, evidence of HIV infection has preceded AIDS by just a few years (CDC. MMWR 1981;30:250; CDC. MMWR 1981;30:305; Jaffe et al. Ann Intern Med 1985;103:210; U.S. Census Bureau; UNAIDS).

Many studies agree that only a single factor, HIV, predicts whether a person will develop AIDS.

Other viral infections, bacterial infections, sexual behavior patterns and drug abuse patterns do not predict who develops AIDS. Individuals from diverse backgrounds, including heterosexual men and women, homosexual men and women, hemophiliacs, sexual partners of hemophiliacs and transfusion recipients, injection-drug users and infants have all developed AIDS, with the only common denominator being their infection with HIV (NIAID, 1995).

In cohort studies, severe immunosuppression and AIDS-defining illnesses occur almost exclusively in individuals who are HIV-infected.

For example, analysis of data from more than 8,000 participants in the Multicenter AIDS Cohort Study (MACS) and the Women’s Interagency HIV Study (WIHS) demonstrated that participants who were HIV-seropositive were 1,100 times more likely to develop an AIDS-associated illness than those who were HIV-seronegative. These overwhelming odds provide a clarity of association that is unusual in medical research (MACS and WIHS Principal Investigators, 2000).

In developing countries, patterns of both rare and endemic diseases have changed dramatically as HIV has spread, with a far greater toll now being exacted among the young and middle-aged, including well-educated members of the middle class.

In developing countries, the emergence of the HIV epidemic has dramatically changed patterns of disease in affected communities. As in developed countries, previously rare, “opportunistic” diseases such as PCP and certain forms of meningitis have become more commonplace. In addition, as HIV seroprevalence rates have risen, there have been significant increases in the burden of endemic conditions such as tuberculosis (TB), particularly among young people. For example, as HIV seroprevalence increased sharply in Blantyre, Malawi from 1986 to 1995, tuberculosis admissions at the city’s main hospital rose more than 400 percent, with the largest increase in cases among children and young adults. In the rural Hlabisa District of South Africa, admissions to tuberculosis wards increased 360 percent from 1992 to 1998, concomitant with a steep rise in HIV seroprevalence. High rates of mortality due to endemic conditions such as TB, diarrheal diseases and wasting syndromes, formerly confined to the elderly and malnourished, are now common among HIV-infected young and middle-aged people in many developing countries (UNAIDS, 2000; Harries et al. Int J Tuberc Lung Dis 1997;1:346; Floyd et al. JAMA 1999;282:1087).

HIV can be detected in virtually everyone with AIDS.

Sensitive testing methods, including the polymerase chain reaction (PCR) and improved culture techniques, have enabled researchers to find HIV in patients with AIDS with few exceptions. HIV has been repeatedly isolated from the blood, semen and vaginal secretions of patients with AIDS, findings consistent with the epidemiologic data demonstrating AIDS transmission via sexual activity and contact with infected blood (Bartlett, 1999; Hammer et al. J Clin Microbiol 1993;31:2557; Jackson et al. J Clin Microbiol 1990;28:16).

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.

12th May 2015

ANOTHER note added in response to Timothy Julian, below, who seems not to understand anything about retrovirus and especially lentivirus evolution.  Here is an unrooted radial relationship diagram (aka “phylogenetic” diagram) depicting whole genome sequence relationships between HIV-1, HIV-2, 2 SIVs, Maedi-Visna ad bovine leukaemia viruses, feline and bovine immunodeficiency and human and simian T-cell lymphotropic viruses.  Done by me today from Genbank sequences, using CLC Genomics Workbench ver 7.

Radial tree for retrovirus complete genome sequences

Radial tree for retrovirus complete genome sequences

What it shows is that:

  • there is a distinctive clustering of HIVs and of SIVs, with MVV as a apparently closer relative than the FIVs, in a cluster of lentiviruses that includes BIV – and I note HIV-1 is more closely related to an SIV than it is to HIV-2, and there are three branches to the H/SIV tree ALL of which are internal to MVV and the FIVs and BIV.
  • the H/STLVs cluster together as relatives, with HTLV-1/2/3 being most closelt related to STLV-1/2/3 – which, seeing as the HTLVs are supposed to have derived from the STLVs, is hardly surprising.
  • BLV is only distantly related to the TLV cluster, as is expected given that it is a leukaemia virus but one of a very different species

If HIV-1 derives from artificial constructs derived from FIVs, which are less closely related to  them than is MVV, then is the same true for the whole primate cluster?  Really?  When it is pretty obvious that they are (a) evolutionarily related most closely to one another, (b) evolutionarily diverged to quite a considerable extent?  So were they all made individually??  Then cleverly given to different bush-dwelling primates in Africa?  How desperately unlikely is that??  You appear not to have heard of teh principle of parsimony, which is that the simplest explanation that covers all of the facts is probably correct – which in this case, is that both HIVs and all of the SIVs have a common evolutionary origin, thousands of years ago – and that all lentiviruses also have a common origin, millions of years ago.

Seriously, Timothy: give it a rest.  You know less than Jon Snow.

Scoop.it: Virology News

11 February, 2012

This is just to announce that I will be regularly posting “Virology News” updates on a new Scoop.it site I have just set up – as well as occasionally updating another Scoop.it site – “Virology and Bioinformatics from Virology.ca” – which is curated by Chris Upton, of Univ Victoria in Canada.

Even more ways to get your daily viral fix…B-)

When dinner could kill you: smoked chimpanzee, anyone?

14 January, 2012

ProMED Mail this morning had a rather alarming item: “BUSHMEAT TRADE, DISEASE TRANSMISSION RISK”.  They reported on a study, highlighted in a BBC report, of possible pathogens imported into the USA via bushmeat from Africa, confiscated at airports.  This in turn derived from a PLoS One paper – “Zoonotic Viruses Associated with Illegally Imported Wildlife Products“, by Kristine Smith et al., published on January 10th 2012.  Their abstract:

The global trade in wildlife has historically contributed to the emergence and spread of infectious diseases. The United States is the world’s largest importer of wildlife and wildlife products, yet minimal pathogen surveillance has precluded assessment of the health risks posed by this practice. This report details the findings of a pilot project to establish surveillance methodology for zoonotic agents in confiscated wildlife products. Initial findings from samples collected at several international airports identified parts originating from nonhuman primate (NHP) and rodent species, including baboon, chimpanzee, mangabey, guenon, green monkey, cane rat and rat. Pathogen screening identified retroviruses (simian foamy virus) and/or herpesviruses (cytomegalovirus and lymphocryptovirus) in the NHP samples. These results are the first demonstration that illegal bushmeat importation into the United States could act as a conduit for pathogen spread, and suggest that implementation of disease surveillance of the wildlife trade will help facilitate prevention of disease emergence.

What was even more horrifying were the pictures of confiscated items – herewith their Figure 1.

doi:10.1371/journal.pone.0029505.g001

It boggles my mind how anyone could even consider smuggling this sort of thing into anywhere – and I am hoping that the US Customs has the same sorts of detection mechanism – as in, well-trained beagles – as they used to have in Miami Airport to detect biltong [dried spiced meat] smuggled in from South Africa.  Those dogs were seriously good – trouble is, they really loved the biltong they got as a reward, too, and it makes my skin crawl rather to imagine a beagle salivating over smoked vervet monkey.

The ProMED post comments further:

No one really knows the scale of the illegal trade in wildlife meat, or bushmeat as it is often called, but a 2010 study estimated that 5 tonnes of the material per week was being smuggled in personal baggage through Roissy-Charles de Gaulle airport in Paris, France. And in addition to the meat products, there is a big trade in live wild animals. Much of this is perfectly legal and supplies the pet industry. Nonetheless, these animals also require improved pathogen surveillance, say the researchers.

One has only to remember that the monkeypox outbreak in the USA in recent times originated in an African animal imported live – see Viroblogy here – to realise the potential danger posed by international movement of wild-caught animals – or even of laboratory animals, as happened in the Ebola Reston incident.

The list of animals from whom parts were found is also rather disturbing: this included chimpanzee, sooty mangabey, and “green monkey” or vervet.  Virologists will not need reminding, but others may, that HIV-1 originated in chimpanzees and HIV-2 in mangabeys – and that although these viruses were not found this time, the PLoS One paper notes:

“Although we did not find SIV or STLV in the limited number of specimens in this study, these viruses have been found in high prevalences in NHP specimens at bushmeat markets and in hunted NHPs [8], [32], [33]. HIV-1 and HIV-2 emerged as a result of several spillover events of SIV from chimpanzees and mangabeys, respectively, that were likely hunted for bushmeat in central and western Africa [30]. Serosurveillance studies have shown thirty-five different species of African NHPs harbor lentivirus infections, with a prevalence of SIV in up to 35% of free-ranging chimpanzees, and 30–60% of free-ranging sooty mangabeys and green monkeys [30], [31], [33], [34].”

So really, it is just a matter of time before meat that contains SIVs or STLVs gets through into the USA and other world centres – and a whole new wave of zoonotic infection could start.  It really is inexcusable that people living in developed countries should be importing meat derived from endangered species in the first place.  It is made worse that developed countries like the USA find it necessary to import LIVE animals as pets – and while the monkeypox outbreak was caught early, the next one may not be.

So forget the “engineered” H5N1 paranoia, folks – be a LOT more scared of the cute rodent in a cage near you, or what your neighbour may be eating….

Virology Africa 2011: viruses at the V&A Waterfront 2

19 December, 2011

We thank Russell Kightley for permission to use the images

Marshall Bloom (Rocky Mountain Laboratories, NIAID) opened the plenary session on Thursday the 1st of December, with a talk on probing the pathogen-vector-host interface of tickborne flaviruses.   Although thoroughly infected with a rhinovirus, he held our attention most ably while reminding us that while many flaviviruses are tick borne, the hard and soft body ticks that vector them are very phylogenetically different – as different as they are from spiders – meaning that if similar flaviruses replicated in them, these viruses may have much wider host range than we know.

He pointed out that while about 95% of the virus life cycle takes place in a tick, transmission to a vertebrate means suddenly adapting to a very different host.  Infection in ticks is persistent, as befits their vector role – but vertebrate infection generally is not.  It was interesting, as a sometime plant virologist, to hear that they look for dsRNA as a marker for replication, and do Ab staining for it: the technique was invented with plant viruses, and very few other virologists seem to appreciate that dsRNA can be quite easily isolated and detected.

They compared Vero and tick cells for virus replication, and saw significant differences: while tick cells could go out to 60+ days and look fine, Vero cells were severely affected at much shorter times post infection.  There was also 100-fold less virus in tick cells, and prominent tubular structures in old infected tick cells.  He noted that ticks evade host defences quite efficiently: eg they suppress host clotting during feeding, and there is huge gene activation in the tick during feeding.  In another study to envy, they are doing array work on ticks to see what is regulated and how.

 Linda Dixon (Institute for Animal Health, Pirbright, UK) recounted her lab’s work on African swine fever (ASFV), a poxvirus-like large DNA virus.  The virus is endemic to much of Africa, and keeps escaping – and there is no effective  vaccine to prevent spread, so regulation is by slaughter.  There are 3 types of isolate, with the most highly pathogenic causing up to 10% fatality and a haemorrhagic syndrome.  She described how in 2007 the virus had spread from Africa to Georgia, then in 2009 to southern Russia and all way to the far north, in wild boar.

There are more than 50 proteins in the dsDNA-containing virion; two infectious forms similar to the poxviruses with multilayer membranes and capsid layers can form, and neutralising Ab play no part in protection as a result.  They studied the interaction of viruses with cells and the immune system, and compared the genomes of pathogenic and non-pathogenic strains, in order to understand how to develop an effective vaccine.

The biggest differences were large deletions in non-virulent isolates, including genes coding for  proteins responsible for binding to RBC, and various immune evasion multicopy genes.  They planned to target regions to delete to make an attenuated virus for vaccine.  They had found non-essential genes involved in immune evasion, and ones that lower virulence, and had been systematically cutting them out.  She noted that pigs can be protected if they survive natural infection and if vaccinated with TC-attenuated virus, and can be protected by passive transfer of Abs from immune pigs – which indicated that an effective live vaccine was very possible.

Subunit vaccines were being investigated, and they had found partial protection with baculovirus-expressed proteins.  They were doing genome-wide screens for protective Ag, and were pooling Ags expressed from predicted ORFs in immunization trials – up to 47 Ags without reduction in specific  T cell responses.

Discovery One

My former labmate Dion du Plessis (Onderstepoort Veterinary Institute, OVI) made a welcome return to Cape Town, with a talk entitled “2011: A Phage Odyssey”.  He explained the title by noting the distinct resemblance of P1 coliphage to the Discovery One spacecraft dreamed up by Arthur C Clarke and Stanley Kubrick – and then went on to exuberantly and idiosyncratically recount a brief history of bacteriophages and their use in biotechnology since their discovery.  A revelation from his talk was that the first discovery of phages was probably described by a gentleman named Hankin, in 1896 in Annales de l’Institut Pasteur: he

The 1896 paper from Annales de l'Institut Pasteur

showed that river water downstream of cholera-infested towns on the Jumma river in India contained no viable Cholera vibrio – and that this was a reliable property of the water.  We were also introduced to the concept of turtles as undertakers in the Ganges….

He took us through the achievements of the Phage Group of Max Delbruck and others – where science was apparently fun, but also resulted in the establishment of modern molecular biology – through to the use of phages as exquisitely sensitive indicators immunochemistry studies in the 1960s.

All too soon we got to the modern uses of phages, with 3 types of gene library – random peptide, fragmented gene, and antibody V regions – being used to make recombinant phage tail proteins to be used for “panning” and enrichment purposes, in order to select either specific antibodies or antigens.  Dion manages a research programme at OVI aimed at developing a new generation of veterinary vaccines – and has for some years now been making significant progress in generating reagents from a chicken IgY single-chain Fv phage display library.

Carolyn Williamson (IIDMM, UCT) gave us an update on CTL epitopes associated with control of HIV-1 subtype C infections.  She said that it was now known that genome-wide association studies (GWAS) gives you certain HLAs which are associated with low viral load, and others with high – meaning that to some extent at least, control of infection was down to genetic luck.  She noted that they and others had shown that CTL escape was quick: this generally happened in less than 5 weeks in acute phase infections.

They had looked for evidence of a fitness cost of CTL escape – and shown that it exists.  She noted that this meant that even if one has “bad” HLA genes, if one was infected with a virus with fitness cost mutations from another, that one could still control infection.

It had been shown that “controllers” mainly have viruses with attenuating mutations, or have escapes in the p24 region – and it was a possible vaccine strategy to include these mutated epitopes in vaccines to help people with infections control their infections.

An interesting topic she broached was that of dual infections – there was the possibility of modelling if infection with two different viruses results in increased Ab neutralisation breadth, and if one would get different results if infections were staggered, possibly with increased nAb evolution if isolates were divergent.  She noted it was possible to track recombination events with dual virus infections too.

It was interesting that, as far as Ab responses went, there were independent responses to 2 variants and one could get a boost in Ab titres to the superinfecting virus, but not a boost to Abs reacting with the originally-infecting virus

Carolyn was of the opinion that HIV vaccines needed to include CTL epitopes where escape is associated with fitness cost.  She also reiterated that superinfection indicated that one can boost novel responses, which I take to mean that therapeutic applications are possible.

Ulrich Desselberger (University of Cambridge) is a long-time expert on rotaviruses and the vaccines against them, and it was a pleasure to finally hear him speak – and that he was mentoring young people in South Africa.  He said that more than a third of children admitted to hospital worldwide were because of rotavirus infections, meaning that the viruses were still a major cause of death and morbidity – and they were ubiquitous.

He reviewed the molecular biology and replication cycle of rotaviruses in order to illustrate where they could be targeted for prevention of infection or therapy, and noted that drugs that interfere with lipid droplet homeostasis interfere with rotavirus replication because 2 viral proteins associated proteins of lipid droplets.

He stated that there were lots of recent whole-genome sequences – we already there were many types, based on the 2 virion surface proteins; we  now know that other genes are also highly variable.  As far as correlates of immunity were concerned, VP7 & 4 were responsible for eliciting neutralising Ab.  Additionally, protective efficacy of VP6 due to elicitation of non-neutralising Ab had been shown in mice – but not in piglets, and not convincingly in humans.  Abs to VP2, and NSP2 and 4 were also partially protective in humans.  It was interesting that protection was not always correlated with high titre nAb responses.

He noted that in clinical disease primary infections partially protected against subsequent infections which are normally milder; subsequently no disease was seen even when infection occurred.  Cross-protection occurred at least partially after initial infection, and this got better after more exposure.  There was evidence one could get intracellular neutralisation by transcytosed Ab, and especially to VP6.  Ab in the gut lumen was a good indication of protection.

As far as the live modern vaccines were concerned, Merck’s Rotateq elicited type-specific nAb, with 9% of recipients shedding live virus.  GSK’s Rotarix gets elicits cross-reactive nAb and one gets 50% of recipients shedding virus.

While the vaccines seemed safe, he noted that where vaccines had been introduced, efficacy ranged from 90% in the USA and Europe, down to as low as 48% in Bangladesh, Malawi and SA, due to type mismatch, and that efficacy was correlated inversely with disease incidence and child mortality generally.  He mentioned that there had been much VLP work, but that none of the candidates was near licensure.

Johan Burger (Stellenbosch University) spoke on one of the more important non-human virus problems in our immediate environment – specifically, those affecting wine grape production in our local area.  He opened by stating that SA now produced 3.7% of the world’s wine, making grapes a nationally and especially locally important crop.  Leafroll disease was a major worldwide problem – as well as being the reason for the wonderful autumn reddening seen in grapevines, it also significantly limited production in affected vineyards.  His laboratory has done a lot of work in both characterising viruses in grapevine, and trying to engineer resistance to them.  Lately they were also investigating the use of engineered miRNAs as a response to and means of controlling, virus infection.

His group has for a couple of years been involved in “metaviromic” or high-throughput sequencing studies of grapevines, with some significant success in revealing unsuspected infections.  In this connection, he and Don Cowan pointed out that they had lots of data that they ignore – but which we should keep and study, as a resource for other studies not yet thought of.

As far as Johan’s work went, novel viruses kept popping up, including grapevine virus E (GVE), which hitherto had only been found in Japan.  They were presently looking at Shiraz disease, which was unique to SA, and was still not understood.  This was infectious, typified by a lack of lignification which led to rubbery vines, and kills plants in 5 years.  It also limits the production of the eponymous grapes – a crime when SA shirazes seem to be doing so well!

Veterinary Virology and Vaccines parallel session.

I again dodged the clinical / HIV session because of my personal biases, and was again treated to a smorgasbord of delight: everyone spoke well, and to time, and I was really gratified to see so many keen, smart young folk coming through in South African virology.  It was also very interesting to see highly topical subjects like Rift Valley fever and rare bunyavirus outbreaks being thoroughly covered, so I will concentrate on these.

P Jansen van Veeren (NICD, Johaanesburg) was again a speaker, this time representing his absent boss, Janusz Paweska.  He gave an account of the 2010 Rift Valley fever outbreak in SA, and epidemiological findings in humans – something of keen interest to me.  He said there had been some forecasting success for outbreaks in East Africa; however, there were long gaps between outbreaks, which were generally linked to abnormal rainfall and movement of mosquito and animal hosts.  RVFV isolates differed in pathogenicity but were structurally and serologically indistinguishable – because virulence was due to the NSs protein, and not a virion component.  He recounted how artificial flooding of a dambo in Kenya resulted in a population boom in the floodwater Aedes mosquitoes responsible for inititating an outbreak, and then of the Culex which maintained the epidemic.  He said there was a strong correlation between viral load and disease severity.

In terms of South African epidemiology, there had been smaller outbreaks from 2008 round the Kruger National Park (NE SA), then in the Northern Cape and KZN in 2009.  People had been infected from autopsy of animals, and handling butchered animal parts.  The 2010 outbreak started in the central Free State after an unusually wet period, and had then spread to all provinces except Limpopo and KZN.  In-house serological methods at the NICD were validated in-house too: these were HAI screening and IgM and IgG ELISAs and a virus neutralisation test.  They had got 1600+ samples of human serum, and confirmed 242 cases of disease and 26 deaths for 2010.

He noted that with winter rains there was a continuous outbreak in the Western Cape, and in 2011 the epidemic had started again in the Eastern and Western Cape Provinces, but has since tailed off.  Some 82% of human cases were people who occupationally handled dead animals, although there was some possibility of transmission by mosquitoes.

In human cases there was viraemia from 2-7 days, with IgM present transiently from 3 days at low level.  They had sequenced partial GP2 after PCR from 47 isolates, and showed some recombination occurring.  The 2010 isolates were very closely related to each other, and to a 2004 Namibian isolate.  There had been no isolation from mosquitoes yet.

Two talks on FMDV followed: Belinda Blignaut (OVI and Univ Pretoria) spoke on indirect assessment of vaccine matching by serology, and Rahana Dwarka (OVI) on a FMDV outbreak in KZN Province in 2011.  Belinda’s report detailed how 6 of 7 serotypes of FMDV occur in SA, with SAT-1 and -2 and O the most common – and that vaccines needed to be matched to emerging strains.  This was done by indirect vaccine matching tests such as serological r-value, determined by the ratio of the reciprocal serum titre to the heterologous virus against that to the homologous virus.  They had put 4 different viruses into cattle and got sera to test a range of 26 newly isolated viruses.  While they had not got sequence from the test panel viruses, indications were that topotype 3 viruses are antigenically more disparate and that a vaccine consisting of topotype 1 or 2 antigens may not be effective in the control of FMD.

In introducing Rahana’s talk, the chair (Livio Heath, OVI) mentioned that there had been 5 different major animal pathogens causing outbreaks in SA over the last 3 years – and that they had to produce reagents and validate tests for ASFV, classical swine fever (CSF) and FMDV, etc, with each outbreak.  Rahana described how they had neutralisation assays and blocking and competition ELISA for FMDV, as well as a big database of isolates from buffalo in KZN – so they were well-placed to type viruses found in cattle in the region.

C van Eeden (Univ Pretoria) had an intriguing account of their investigation of the occurrence of an orthobunyavirus causing neurological symptoms in horses and wildlife.  Horses seem to be particularly vulnerable to many of the viruses involved in such disease, and so are a useful sentinel species.  Shuni virus was first isolated from Culicoides midges and sheep and a child in Nigeria in the 1960s.  SA workers subsequently found it in some livestock and Culex mosquitoes and in horses.  The virus was shown to be a neurologic disease agent in horses and wildlife – then disappeared for some 30 years, much like Ebola.  There is apparently a new research unit at UP with a BSL3 lab, so they are well equipped to do tests with the virus.

Ms van Eeden noted that the incidence of encephalitic disease in humans and animal in SA is underreported, and the causes are mainly unknown – a revelation to me!  Horses are susceptible to many of the agents, and are useful sentinels – workers have identified flavi- and alphaviruses in some outbreaks, but many are not IDed.  They had done cell culture and EM on samples from an ataxic horse: they got a bunyavirus-like virus by EM, and did bunya-specific PCR, and got Shuni virus back.  Sequence relationships showed no linkage to type of animal or date, in subsequent samplings from horses, crocodiles,  a rhino and a warthog, and from blood, brain and spinal cord.  All positive wildlife were sampled in Limpopo Province; horses only from most other provinces.

She noted that latest cases were neurological, whereas previously these were mainly febrile.  The virus accounted for 10% all neurological cases, with a 50% fatality rate.  She noted further that vets often work without masks or gloves, and so had no protection from exposure in such cases….  There was no idea on what the vector was, but they would like to test mosquitoes, etc.  Ulrich Desselberger suggested  rodents may be a reservoir, but they don’t know if this is true.

Stephanie van Niekerk (Univ Pretoria) investigated alphaviruses as neurological disease agents in African wildlife.  The most common alphaviruses in SA are Sindbis and Middelburg viruses.  Old World alphaviruses are usually not too bad, and cause arthritic and febrile symptoms, while New World cause severe neurological diseases.  Sindbis was been found in SA outbreaks in 1974.  However, Stephanie noted that a severe neurological type had appeared since 2008 in horses.  Accordingly, they looked at unexplained cases in wildlife in the period 2009-2011: brain and spinal cord samples were investigated for all cases.  They found alphavirus in a number of rhinos, buffalo, warthog, crocodiles and jackal – and all except for one rhino were Middelburg virus.  They want to isolate viruses in cell culture, and increase the size of regions used for cDNA PCR.  Stephanie said the opinion was that the values of the animal involved justifies the development of vaccines.

 

Virology Africa 2011: viruses at the V&A Waterfront 1

12 December, 2011

We thank Russell Kightley for permission to use the images

Anna-Lise Williamson and I again hosted the Virology Africa Conference (only the second since 2005!), at the University of Cape Town‘s Graduate School of Business in the Victoria & Alfred Waterfront in Cape Town.  While this was a local meeting, with just 147 attendees, we had a very international flavour in the plenaries: of 18 invited talks, 9 were by foreign guests.  Plenaries spanned the full spectrum of virology, ranging from discovery virology to human papillomaviruses to HIV vaccines to tick-borne viruses to bacteriophages found in soil to phages used as display vectors, and to viromes of whole vineyards.  There were a further 52 contributed talks and 41 posters, covering topics from human and animal clinical studies, to engineering plants for resistance to viruses.

A special 1-day workshop on “Human Papillomaviruses – Vaccines and Cervical Cancer Screening” preceded the main event: this was sponsored by Merck Sharp & Dohme, Roche and Aspen Pharmacare, and had around 90 attendees.  Anna-Lise Williamson (NHLS & IIDMM, UCT) opened the workshop with a talk entitled “INTRODUCTION TO HPV IN SOUTH AFRICA – SCREENING FOR CERVICAL CANCER AND VACCINES”, and set  the stage for Jennifer Moodley (Community Health Dept, UCT) to cover health system issues around the prevention of cervical cancer in SA, and the newly-minted Dr Zizipho Mbulawa (Medical Virology, UCT) to speak on the the impact of HIV infection on the natural history of HPV.  This last issue is especially interesting, given that HIV-infected women may have multiple (>10) HPV types and progress faster to cervical malignancies, and HPV infection is a risk factor for acquisition of HIV.  The Roche-sponsored guest, Peter JF Snijders (VU University Medical Center, Amsterdam), gave an excellent description of novel cervical screening options using primary HPV testing, to be followed by two accounts of cytological screening in public and private healthcare systems in SA, by Irene le Roux (National Health Laboratory Service) and Judy Whittaker (Pathcare), respectively.  Ulf Gyllensten (University of Uppsala, Sweden) described the Swedish experience with self-sampling and repeat screening for the prevention of cervical cancer, especially in groups that are not reached by standard screening modalities.  Hennie Botha and Haynes van der Merwe (both University of Stellenbosch) closed out the session with talks on the effect of the HIV pandemic on cervical cancer screening, and a project aimed at piloting adolescent female vaccination against HPV infection in Cape Town.

The next part of the Workshop overlapped with the Conference opening, with a Keynote address by Margaret Stanley (Cambridge University) on how HPV evades host defences (sponsored by MSD), and another by Hugues Bogaert (HB Consult, Gent, Belgium)) on comparisons of the cross-protection by the two HPV vaccines currently registered worldwide (sponsored by Aspen Pharmacare). Margaret Stanley’s talk was a masterclass on HPV immunology: the concept that such a seemingly simple virus (only 8 kb of dsDNA) could interact with cells in such a complex way, was a surprise for all not acquainted with the viruses.  Bogaert’s talk was interesting in view of the fact that the GSK offering, which has only only two HPV types, raises far higher titre antibody responses than the MSD vaccine with four HPV types, AND seems to elicit better cross-protective antibodies: this should help inform choice of product from the individual point of view.  However, the fact that MSD seems able to respond better to national healthcare system tenders in terms of price per dose is also a major factor in the adoption stakes.

The Conference proper started with a final address by Barry Schoub, long-time but now retired Director of the National Institute of Virology / National Institute of Communicable Diseases in Johannesburg, and also long-time CEO of the Poliomyelitis Research Foundation (PRF): this is possibly the premier funding agency for anything to do with viruses in South Africa, and a major sponsor of the Conference.  He spoke on the history of the PRF, and how it had managed to shepherd an initial endowment of around 1 million pounds in the 1950s, to over ZAR100 million today – AND to dispense many millions in research project and bursary funding in South Africa over several decades.

The first session segued into a welcoming cocktail reception and registration at the Two Oceans Aquarium in the V&A Waterfront: this HAS to be one of the only social events for an academic conference where the biggest sharks are the ones in the tank, and not in the guest list!  I think people were suitably blown away – as always, in the aquarium – and the tone was set for the rest of the meeting.  The wine and food were good, too.

The first morning session of the conference featured virus hunting and HIV vaccines, as well as plant-made vaccines and more HPV.  W Ian Lipkin (Columbia University, USA) opened with “Microbe Hunting” – which lived up to its title very adequately, with discussion of a plethora of infectious agents.  As well as of the methods newly used to discover them, which include high-throughput sequencing, protein arrays, very smart new variants on PCR….  I could see people drooling in the audience; the shop window was tempting enough to make one jump ship to work with him without a second thought.  He said that probably 99% of vertebrate viruses remain to be discovered, and that advances in DNA sequencing technology were a major determinant in the rapidly-increasing pace of discovery.  He made the point that while the emphasis in the lab had shifted from wet lab people to bioinformatics, he thought it would move back again as techniques get easier and more automated – meaning (to me) that there is no substitute for people who understand the actual biological problems.  It was interesting that, while telling us of his work on the recently-released blockbuster “Contagion” – where “the virus is the star!” – he showed a slide with a computer in the background running a recombination detection package called RDP, which was designed in South Africa.  It can also be seen in the trailer, apparently.  Darren Martin will not be looking for royalties or screen credits, however.

Don Cowan (University of the Western Cape) continued the discovery theme, albeit with bacteriophages as the target rather than vertebrate viruses.  It is worth emphasising that phages probably represent the biggest source of genetic diversity on this planet – and given how even the most extreme of microbes have several kinds of viruses, as Don pointed out, it is possible that this extends to neighbouring planets too [my speculation – Ed].  He occupies an interesting niche – much like the microbes he hunts – in that he specialises in both hot and cold terrestrial desert environments, which are drastically understudied in comparison to marine habitats.  He made the interesting point that metagenome sequencing studies such as his own generate data that is in danger of being discarded without reuse, given that folk tend to take what they are interested out of it and neglect the rest.

Anna-Lise Williamson (NHLS, IIDMM, UCT) then described the now-defunct SA AIDS Vaccine Initiative vaccine development project at UCT.  It is rather sobering to revisit a project that used to employ some 45 people, and had everything from Salmonella, BCG, MVA, DNA and insect cell and plant-made subunit HIV vaccines in the pipeline – and now employs just 5, to service the two vaccines that made it into into clinical trial.  The BCG-based vaccines continued to be funded by the NIH, however, and the SA National Research Foundation funds novel vaccine approaches.  Despite all the funding woes, the first clinical trial is complete with moderate immunogenicity and no significant side effects, and two more are planned: these are an extension of the first – HVTN073/SAAVI102 – with a Novartis-made subtype C gp140 subunit boost, and the other is HVTN086/SAAVI103, which compprises different commbinations of DNA, MVA and gp140 vaccines.

It was clear from the talk that if South Africa wants to support local vaccine development, the government needs to support appropriate management structures to enable this – and above all, to provide funding.  However, all is not lost, as much of the remaining expertise in several of the laboratories that were involved in the HIV vaccine programme can now involve themselves in animal vaccine projects.

Plant-made HPV16 VLPs

Ed Rybicki made it an organisational one-two with an after-tea plenary on why production of viral vaccines in plants is a viable rapid-response option for emerging or re-emerging diseases or bioterror threats.  The talk briefly covered the more than 20 year history of plant-made vaccines, highlighting important technological advances and proofs of concept and efficacy, and concentrated on the use of transient expression for the rapid, high-level expression of subunit vaccines.  Important breakthoughs that were highlighted included the development of the Icon Genetics TMV-based vectors, Medicago Inc and Fraunhofer USA’s recent successes with H5N1 and H1N1 HA protein production in plants – and the Rybicki group’s successes with expression of HPV L1-based and E7 vaccine candidates.  The talk emphasised how the technology was inherently more easily scalable, and quicker to respond to demand, than conventional approaches to vaccine manufacture – and how it could profitably be applied to “orphan vaccines” such as for Lassa fever.

Ulf Gyllensten had another innings in the main conference, with a report on a study of a possible linkage of gene to disease in HPV infections – which could explain why some people clear infections, and why some have persistent infections.  They used the Swedish cancer registry (a comprehensive record since the end of the 1950s) to calculate familial relative risk of cancer of the cervix (CC): relative risk was  2x for a full sister, the same for a mother-daughter pair and the risk for a half sister was 50% higher while risk was not linked to non-biological siblings or parents, meaning the link was not environmental.  A preliminary study found HLA alleles associated with CC, and increased carriage of genes was linked to increased  viral load.   A subsequent genome wide association study using an Omni Express Bead Chip detecting700K+ SNPs yielded one area of major interest, on Chr 6 – this is a HLA locus.  They got 3 independent signals in the HLA region and can now potentially link HPV type and host genotype for a prediction of disease outcome.  Again, the kinds of technology available could only be wished for here; so too the registry and survey options.

Molecular and General Virology contributed talks parallel session

I attended this because of my continued fascination with veterinary and plant viruses – and because Anna-Lise was covering the Clinical and Molecular session – and was not disappointed: talks were of a very high standard, and the postgraduate students especially all gave very good accounts of themselves.

Melanie Platz (Univ Koblenz-Landau, Germany) kicked off with a description of a fascinating interface between mathematics and virology for early warning, spatial awareness and other applications.  She gave an example using a visual representation of risk using GIS for Chikungunya virus, based on South African humidity and temperature data going back nearly 100 years: this had a 3D plot model, into which one could plug data to get predictions of mosquito likelihood.  They could generate risk maps from the data, to both inform public and policy / planning.  They had a GUI for mobile devices for public information, including estimates of risk and what to do about it, including routes of escape.

Cover Illustration: J Virol, October 2011, volume 85, issue 20

This was followed by one of my co-supervised PhD students, Aderito Monjane – who recently got the cover of Journal of Virology with his paper on modelling maize streak virus (MSV) movement and evolution, so I will not detail more here.  However, even as a co-supervisor I was blown away by the fact that he was able to show animations of MSV spread – at  30 km/yr, across the whole of sub-Saharan Africa.

Christine Rey of Wits University provided another state-of-the-art geminivirus talk, with an account of the use of siRNAs and derivatives for silencing cassava-infecting geminiviruses.  They were using genomic miRNA precursors as templates to make artificial miRNAs containing viral sequences, meaning they got no interference with nuclear processing and there was less chance of recombination with other viruses, a high target specificity, and the transgenes would not be direct targets of virus-coded suppressors.  They could also use multiple miRNAs to avoid mutational escape.  The concept was successful in tobacco, and they had got transformation going well for cassava, so hopes were high for success there.

Dionne Shepherd (UCT) spoke on our laboratory’s 15+ year work on engineered resistance in maize to MSV.  She pointed out that the virus threatens the livelihood of 200 million+ subsistence farmers in Africa, and is thought to be the biggest disease concern in maize – which is still the biggest edible crop in Africa.  Most of the work has been described elsewhere with another journal cover; however, new siRNA-based constructs still under investigation were even more effective than the previous dominant negative mutant-based protection: the latter gave 50-fold reduction in virus replication, but silencing allowed > 200-fold suppression of replication.

2-colour surface rendition of HcRNAV

Arvind Varsani – a former UCT vaccinology PhD who is now a structural biology and virology lecturer at Univ Christchurch (NZ) – described what is probably the first 3D structure of a virus to come out of Africa.  This was of a 30 nm isometric ssRNA virus – Heterocapsa circularisquama RNA virus (HcRNAV) – infecting a dinoflagellate, which is one of the most noxious red tide bloom agents and is a major factor in killing farmed oysters.  The virus apparently controls the diatom populations.  There are two distinct strains of virus, and specificity of infection is due to the entry process, as biolistic bombardment obviates the block.  The single capsid protein probably has the classic jelly-roll β-barrel fold, but they observe a new packing arrangement that is only distantly related to the other ssRNA (+) virus capsids known.  They will go on to look at structural differences between strains that change cell entry properties.

FF Maree from the Onderstepoort Veterinary Institute and the Univ Pretoria spoke on structural design of FMDV to improve vaccine strains: they wished to engineer viruses by inserting the cell culture adapted HSPG-binding signature sequence and to mutate capsid residues to increase the heat stability of SAT-2 subtype virus vaccines.  If they put the signature sequence in a SAT1 virus, they found it could infect CHO cells – which do not express any of 4 integrins that FMDV binds to, but are far better for large-scale production of the virus than the BHK cells used till now.  It was also possible to increase hydrophobic interactions in the capsid by modeling: eg a VP2 Ser to Tyr replacement gave a considerably better thermal inactivation profile to the virus.

Daria Rutkowska (Univ Pretoria) detailed how African horsesickness orbivirus (AHSV) VP7 protein had significant potential as a scaffold that could act as a vaccine carrier.  The native protein formed as trimers assembled in a VP3+VP7 “core” particle; however, the VP7 when expressed alone could form soluble trimmers – and the “top” domain hydrophilic loop can tolerate large inserts.  The group had very promising FMDV P1 peptide responses from engineered VP7 constructs, including protection of experimental animals.

P Jansen van Veeren of the National Institute of Communicable Disease in Johannesburg finished off the session, with a description of the cellular pathology caused by Rift Valley fever bunyavirus (RVFV) in mice in acute infections.  The virus seems to have been of particular international interest recently as a potential bioterror agent; however, global warming is also responsible for its mosquito vector spreading outside of its natural base in Africa to the Arabian peninsula, and there are fears of the virus getting into Europe soon.  While there are vaccines against the virus, including a live attenuated version, none are licenced for human use.  It was interesting to hear that the viral NP appears to be the main immunogen, as there are massive amounts of NP produced in infection, and huge responses to it in infected animals – and NP immunisation protects mice.  There is a good Ab response but it is not neutralising, while NP is released independently of other proteins from infected cells.  The liver is the major target of virus infection, with a bias to apoptosis of hepatocytes and severe inflammatory responses.  Viral load is linked to these effects and is much lower in vaccinees.  Immunisation reduces liver replication markedly; that in the spleen less so.  A screen of cytokines and other gene responses showed a big down-regulation of many genes in non-vaccinated mice to do with cytokines, and down-regulation of B and T cells and NK cells.  He thinks recombinant vaccine candidates should have both the surface glycoproteins and the NP in order to be effective – and that there is a major need for proper reagents for big animal studies.

HIV Vaccines From Bangkok – 4, and final….

22 September, 2011

Thursday morning started with three parallel oral sessions – and I chose Symposium 07, Characterization of Breakthrough Viruses.  The second talk – by Morgane Rolland, in the US Military HIV Research Program – detailed a study of the sieve analysis of breakthrough viruses in the RV144 Thai trial.  They wished to see whether or not the vaccine could block infection of specific variants, and thought they might see that viruses in vaccinees were evolutionarily distant from the insert in the vaccine, relative to the placebo arm.

HIV and its life cycle

The saw no differences in virus diversity over 10 sequences per person, in 121 people,  71 of whom were in the placebo arm.  They did note, however, that linked transmissions showed less diversity in the env gene than normal – 1 vs 10%.  Over 75% of cases had a single founder virus, in both placebo and vaccine arms.  There was no significant divergence from the vaccine sequence in either group in anything but the Pro aa sequence – with some non-significant evidence for Env variation.

When they looked for Env sites under selection in gp120, they saw 4 in the placebo group at positions 181, 208, 327 and 359 – with less variation in vaccine than placebo recipients.  Rolland speculated that this could be to do with entry being more restrictive in vaccinees?  4 different sites in the vaccine group were under selection: they found that for MHC I epitopes there was a greater distance for vaccine than placebo groups, with a result that was not significant for MHC II epitopes.

There was a trend toward longer Env V2 loop sequences in vaccinees at later times, and a reduced number of cysteines in Env among vaccinees – this was seen also in the VAX004 trial.

Phil Berman – formerly of VaxGen, which made the gp120 for RV144 and earlier trials – mentioned that there was lower variance in Env than in the unsuccessful VAX 003 trial.  Jerome Kim noted that men seroconverting had a much higher incidence of HCV infection – which could be associated with undeclared IV drug use.

Katharine Barr of Univ Alabama spoke next, on the increased incidence of multiple variant transmission of HIV in VAX003 injection drug users.  She noted that this efficacy trial was of gp120 in IV drug users, while VAX004  was in MSM and high-risk women: they speculated that differences if any could be due to transmission route – as in, IV route vs sexual.  She further noted that in RV144, the best (non-significant) effect was in low-risk heterosexuals.

Something that was a little disturbing to me, given HIV transmission in our part of the world is overwhelmingly by heterosexual sex, was that the IV route is responsible for 10% of world infections.  They had looked at transmitted founder viruses – the ones going in and replicating in recipients.  They predicted that consensus of a low diversity lineage is the sequence of the founder virus – and that several founders would give multiple low variance lineages.

She noted that 80% of heterosexual infections are established by single viruses, so there exists a window of opportunity of viral vulnerability when vaccine-induced immunity could block infection.  However, with MSM, the multiple infection goes up to 40%; while injection drug users (IDUs) are less studied, multiplicity goes up  60% in one study and 31% in another….

Looking at Vax003 results, they had asked how high a barrier there had been for placebo infections, and whether in vaccinees there were more or fewer founder viruses?  While they had found that there was an 44% incidence of multiple variant transmission in the  placebo arm, and  22% in the vaccinees, this was unfortunately not significant, given the low numbers.  There was a median of 1.8 viruses per transmission vs 1.3, but this too was not significant.  However, it could mean there is a higher bar for vaccine protection among IDUs, which has important implications for which groups to use in vaccine trials.

Katherine incidentally gave the best answer yet heard to a long and detailed question: “I think that’s a really good question but I have zero data to address it…” = I don’t know.

Which prompted thoughts of new conference drinking games: take a shot every time you hear a speaker say “I would like to thank the organisers for inviting me…”, or “Our hypothesis [generally pronounced hy-PAH-the-sis] was…”, or a question which starts with either “…really good talk / great data” or “So – ummmm – when you/we did…”.

Paul Edlefsen (Fred Hutchinson Cancer Res Ctr) described a sieve analysis of RV144 [and started: “So…umm…” = another shot!].  He repeated the finding that observed correlates of risk generated two hypotheses; namely, that high IgG response to Env protected from HIV infection while a high IgA response interfered with protection.  Additionally, analysis of the antibody response using scaffold V region showed that a high V2 response correlated with a lower infection rate.  He noted that the STEP trial results showed a distinct difference in Gag between vaccine and placebo groups.  He noted further that were only 110 usable subjects in RV144, so they could only detect large sieve effects in their study of CTL and Ab epitope responses.

MUCH MIND-NUMBINGLY BORING STATISTICAL METHODOLOGY FOLLOWED…sorry, Paul!

There were 2 sites of evidence for sieving – aa positions 169 and 181 in the Env V2 loop, in the middle of a region identified by Ab binding array data.  There was also some evidence of covariation among pairs of aa residues in the V2 loop for vaccinees only.

After a long and complicated structural question, he gave the second-best answer of the conference: “I could say that I do, or that I don’t – but I have so little expertise in this area…(laughter)”.  And after long rambling statement: – “I’m sorry, was there a question in there?”

Brandon Keele (National Cancer Inst, MD) described work on NHPs which they had extended to studying human transmission of HIV, on transmitted/founder viruses.  NHP studies show multiple founders because doses are high generally, in order to get 100% infection rates.  One study using very low dose multiple intrarectal exposures to see if one can immunise macaques showed that one virus could do it.  Animals followed up from early times stayed with one evolving variant.

He noted that the consensus sequences in humans posited to have had one transmitted variant are average in  neutralisation susceptibility.  These viruses are all functional in vitro and in vivo and one can get full length viral clones ex NHPs which recap original founder viral load and pathogenicity.  All such viruses use the CCR5 coreceptor.  All HIV clones replicate in CD4 T-cells but not in  macrophages.  The transmission signature is to increase Env processing and infectivity.

They now mix cloned viruses with tags so can follow them in NHP challenge experiments, as most challenge studies have used virus with <1% diversity, which represents a clone in any one epitope – which he felt to be non-reflective of the real world .

The closing plenary session was opened by IAVI‘s Wayne Koff, who remarked that he had heard someone say “The  airport….”, in answer to the session name “Where do we go next?”….

Jeffrey Boyington (Vaccine Res Ctr) described some very impressive work on using structure of Env for rational immunogen design, specifically to target the CD4 binding site as a good target for broadly neutralising Ab.  They used crytallographic data to make proteins best mimicking the struc and then used them as immunogens.  They had used stabilised resurfaced gp120 with mutations around the binding site and isolated dozens of Abs with them from several infected subjects.  Part of the process involved stabilising flexible regions by bolstering cysteine content, removing glycans from the site of interest and adding them to immunodominant sites, and using Chikungunya virus VLPs to multimerise spike proteins for maximal immunogenicity.  Boyington noted that there were 80 native trimers on the surface of the VLPs, and that one can put the Outer Domain of gp120 on the tip of each monomer.  They get good Ab back for gp120 and get CD4 binding site Ab in rabbits.  In rhesus monkeys primed with gp140 trimers they got good boosting and better Abs to the CD4 BS.

Altogether a very impressive account – and one which advances to possibility of other opportunities for the design of other good broad-binding vaccine epitopes.

Rick King of IAVI followed, with an account of the current status and future directions of vector-based HIV vaccines.  He stated that most HIV vaccines now involve vectors – so there is a wealth of data that can be efficacious, so how to use it?  He thinks that we want the next generation of vectored vaccines to block infection and control virus load – meaning a combination of Ab and cellular responses.He noted that in NHPs, SIV protection is possible, and that it requires Env in the vaccine – and that the mechanism of protection is under intense investigation right now.

He further noted that in a DNA prime MVA boost vaccine regime, protection is associated with the avidity of the Abs.  Thus, a major goal is to improve the response to Env, by identifying the nature of the protective response, and enhancing and using native Envs to do it.  He stated in this context that there were only two vaccine regimens using native spike protein – and that one of them is the SA AIDS Vaccine Initiative (SAAVI) vaccine.

It was possible to engineer Env to bind a broader array of broadly neutralising Ab and to incorporate it into vesicular stomatitis virus (VSV) instead of the native G protein spike, or into canine distemper virus (CDV, a measles relative), which replicates in lymphoid tissue.  One could also bias processing of Env in CDV to get better cleavage and presentation.  The rCDV could be put into ferrets and shown to replicate.

He said that while the RV144 vaccine did not control viral load, vaccines can control SIV replication, so we need to have those components in HIV vaccines.  For instance, recombinant live cytomegalovirus (CMV) expressing the whole proteome of SIV could control the virus, this was associated with CD8 effector memory T-cells.

He thought we need to capitalise on information on mechanisms of control, and to increase immunity by use of replicating vectors and heterologous prime/boost combos, and deal with diversity by broadening the response.  The reason for replicating vectors was because live attenuated virus works for SIV – preventing infection and controlling replication.  Possibilities were vaccinia, measles, VSV, Sendai, CMV, AdV, CDV and VSV-HIV chimaeras.  As for diversity, one could increase the number of epitopes by using mosaics, and direct responses using conserved epitopes, as Tomas Hanke has demonstrated in IAVI-funded trials using chimpanzee Ad as prime then MVA as a boost with his HIVCONS Ag.

Finally, there was what I consider to have been the best talk of the conference – simply because it was much wider in scope than the rest: Steven Reed of the Infectious Disease Res Inst, Seattle, described new generation adjuvants for use with HIV.  He started by noting that adjuvants were necessary for lots of things; eg: for T-cell vaccines for TB and leishmania; for Ab response-broadening (Cervarix, HPV vaccine); Ag dose sparing (eg flu); to combat immune sensescence, and for vaccine therapy.

They had focused on a toll-like receptor (TLR4) agonist as an adjuvant, following work that showed that the well-known MPL was a TLR4 agonist ,and vaccines including TLR agonists had been used unknowingly since 1885.

He thinks the ideal adjuvant should have no effect on lymphocytes, no systemic effects, no non-specific B or T cell responses, should elicit potent long-lived responses, should redirect ongoing immune responses, and should be safe and effective in all age groups.  They had accordingly designed GLA – based on lipid A – to bind TLR4: this was purely synthetic, and induces Th1 CD4 helper cells and a broad humoral immunity.  They used a hexaacyl chain length that was preferred by human TLR4, which is restricted to macrophages and dendritic cells, has transient local effects, and reduces inflammation so as to get better central memory.

They can also formulate it differently for different vaccines and can get very different effects thereby.  For example, emulsion alone stimulates Th2 responses while GLA stimulates Th1 even in combo with an emulsion, which helps in leishmania and TB vaccines.

He noted that alum-based adjuvant stimulated mainly a Th2 response, while adding GLA gives a Th1 response with the same antigen.  They get good Ab diversity with GLA and expansion of it with the malaria vaccine – and Ab diversity leads to better neutralisation (eg transl med 2011).

GLA increases and broadens the haemagglutination-inhibtion (HAI) Ab response to the influenza vaccine Fluzone, which contains lots of inactivated virions.  He noted one gets a better protective response against “drifted” viruses – which have evolved away from the vaccine strains – with GLA.  Baculovirus-made H5N1 vaccine requires 30x less vaccine to get the same response with GLA.

It is also possible to get mucosal immunity by IM vaccination with HIV gp140, according to Robin Shattock’s results.

Reed noted that intradermal adjuvants are very rare – and that this looks good with flu vaccines delivered this way.  They were in the process of optimising the adjuvant formulation for intradermal delivery to increase vaccine potency, get mucosal immunity, and CD8+ T-cell responses.  Dermal dendritic cells have a wider range of TLRs than Langerhans cells – so Sanofi target them with ID delivery, and GLA works well to amplify the response.  It was impressive that they could protect ferrets with a single ID vaccine shot of flu vaccine.  It was also interesting that they are working with Medicago Inc., who have one of the most successful plant-produced influenza virus vaccine candidates, presently in human trial.

Thereafter, closing remarks from the conference organiser were as one would expect; people were honoured for their present and long-term contributions – notably Jose Esparza – and the venue of the next conference was announced to be Boston, with Dan Barouch as Organising Chair.

It was a good conference, with all of the high-intensity interactions and presentations one would expect from such a loaded topic.  However, it possibly suffered from over-emphasis of the “RV144 results” – which weren’t that impressive, in my opinion – as part of an effort to keep up perceived momentum from announcement of the RV144 success (small as it was) from the previous meeting.  For me, the highlights were the envelope antigen design talks, and what I managed to catch of the actual virology, and especially analysis of diversity by massively parallel sequencing.

We still don’t have an effective HIV vaccine – but we’re getting closer. 

HIV Vaccines From Bangkok – 3

20 September, 2011

HIV: a retrovirus. Courtesy of http://www.rkm.com.au

The Wednesday morning agenda for the conference followed a somewhat bemusing Tuesday evening entertainment: one day I will learn NOT to involve myself in anything that involves getting onto a fleet of buses in the company of several hundred other people, and especially not in Bangkok!  It took us one-and-a-half HOURS to go from the venue to the Navy Yard for a reception and supper – but the first half an hour was spent just going around the block, such is the traffic density at rush hour.  There followed the standard fare for a conference in any country with any sort of culture: local entertainment (drummers and folk running around with bolts of cloth in this case), together with so-so food with very little choice, too much noise, and no possibility of being heard more than one person away.  But thankfully, only a twenty minute ride back!

“New Prevention Strategies” was the theme for the second set of plenaries – which were opened (unexpectedly; she was second on the programme, but No 1 overslept) by our very own Carolyn WIlliamson (IIDMM, UCT), speaking on implications for combination prevention strategies from HIV pre- and post-infection studies.  Carolyn noted again a point first raised by Pontiano Kaleebu on the first evening, that future vaccine efficacy trials should as as a matter of ethics offer preventions – eg ARVs – as a minimum standard of care, which will affect size and expense as well as endpoints like acquisition and disease progress.

She pointed out that 80% of infections with HIV were due to single viruses – but 20% were due to multiple infections, influenced by dose, IV injection, MSM transmission, inflammatory genital tract infections and the like.  The lesson from study of the Phambili and STEP trial breakthrough infections by sieve analysis showed vaccination had had a selective effect on T-cell pressure.  Phambili got 277 sequences from 43 people, in vaccine and placebo arms.  The Merck vaccine had no effect on the transmission bottleneck.  Scanning sites across the genome showed 2 sites of selection in Gag and 1 site in Nef were significantly different in the two arms; one in the region p6 of Gag looks like an epitope escape.  There was a weaker signal in Phambili than in STEP, however: this was due in part to the lower number of participants (the trial was stopped before recruitment was complete), the fact that there were men and women involved vs mainly men in STEP, among other factors.

It is worth remembering that the Phambili and STEP trials were stopped in 2007, and reported on at a very gloomy AIDS Vaccine Conference in Cape Town (covered here in ViroBlogy).

There was also no effect of pre-exposure Tenofovir on the transmission bottleneck, or evidence of immunity in highly exposed uninfected individuals using the gel – despite the evidence for “chemovaccination” in macaques, due to abortive infection checked by ARVs in target cells.  Thus, chemovaccination did not enhance the of impact microbicides and preinfection immune responses would not interfere with vaccine monitoring by this assay.

Tenofovir did impact early Ifn-g Gag-specific CD4+ responses post infection – indicating that possibly the drug prevents the initial destruction of CD4 cells in the gut, which would be a very valuable result.

Carolyn finished by noting that the implication for combination of preventive therapies is that it will increase the complexity of trials, make them cost considerably more, and make them longer.  However, microbicides that reduce inflammation may dramatically reduce infections, ARVs may increase the barrier to infections, and also increase the time for the effects of vaccination to kick in and increase post infection immunity, and combination of multiple partially effective interventions may have significantly greater impact than any alone.

Helen Weiss (London School of Tropical Medicine and Hygeine) was the late riser: she spoke on lessons from male circumcision for other prevention strategies.  It was interesting to many of us that it was a study in Nairobi in 1989 that showed the effect first – circumcision protected to some extent against in infection even in the presence of genitourinary diseases (GUDs).  A metastudy combining 15 studies subsequently showed reduced risk in all, to a 60% protection level.  Accordingly, three studies had been set up in Uganda, Kenya and SA  in 2005-2007 to directly study the effect.  They saw 50% efficacy in all locations, and all were stopped early as there was an obvious effect, with all participants being offered circumcision.  The studies saw an overall 58% protective effect, and the  effect in the Uganda trial persists up to 5 years post trial.

As for why this should be, Helen said that some studies say that the inner foreskin has a greater density of Langerhans and T-cells compared to the outer – and there is some evidence the inner is more easily infected in explant studies.  HIV infections also induce retention of Langerhans cells within the epidermis of the inner foreskin.  There is evidence that the inner foreskin facilitates efficient entry and translocation of cell-associated HIV, retention of Langerhans cells, and the incidence of infection is greater in men with larger foreskin area.

The conclusion was that one should offer circumcision in HIV prevention studies where heterosexual contact is the mode of transmission.  Among MSM, habitual penile inserters show some effect of protection, while habitual accepters are obviously not protected.

She closed by commenting that scaling up circumcision to 80 % coverage of adults and newborns by 2014 could save US$ 40 billion US: however, the reality was that uptake was slower than planned, with only 2.6% done by 2010.  However, there was obvious buy-in with a fourfold increase in circumcisions between 2009-2010.

While I thought she oversold the intervention rather – it is decidedly less simple than drug or microbicide interventions after all, benefits only one partner directly, and the lesson in South Africa is that even communities with a high circumcision rate can have very high prevalences of HIV infection – there is no doubt that circumcision in combination with pre- and post-exposure ARVs and microbicides cannot do other than have an additive effect in protection, and possibly even a synergistic one in some cases.

For me that was the morning; I missed three very worthy parallel late morning oral sessions while dealing with nagging emails – but started fresh again in the post-lunch period (an aside: best conference coffee break munchies and light lunches I have ever seen…B-), with Oral Session 11 – Mucosal Immunity.

Anthony Smith introduced us to a fascinating study of transcriptional “imprints” correlating with protective immunity in macaques following vaccination with the live attenuated SIV-∆-Nef virus, done by microarrays on RNAs from the cervix.  Smith noted that live attenuated viruses offer some of the best protection available in monkeys, and that SIV Mac239-∆-Nef was one of the best.  They isolated total RNA ex the cervix of rhesus macaques post-challenge with a heterologous virus at 140 days with native virus and tested unvaccinated and vaccinated samples with an Affymetrix rhesus chip.  There was 103-fold less viral RNA in vaccinated animals, and very little overlap of gene expression – only 1% (eg 5 genes) – of 405 vs 246 unvaccinated to vaccinated samples.  There was greater expression of inhibitors of innate immunity and inflammation in vaccinees; MIP3alpha expression was higher in unvaccinated monkeys – this brings in effector cells, including CD4+ T-cells, which would enhance infection.  Unvaccinated monkeys get a signalling cascade of cytokines which cause an inflammatory response – vaccinees get a short circuit in this signalling by mucosal conditioning with mutant virus.  There were important differences in humoral responses too, which were not reported here.  In light of this one could almost wish that the proposed trial in humans in the 1990s of the natural Nef deletant HIV-1 found in Sydney and associated with long-term non-progression had gone ahead – but only almost, as people with the virus did eventually start to progress to AIDS.

Steve Reeves then spoke on mucosal natural killer (NK) cells in SIV infected monkeys in chronic infection: he noted that NK cells respond early in infection in a variety of tissues.  They act to suppress viral replication in vitro, and are linked to disease control in vivo.  while much of what he said was straight over my head – I really do not have much truck with cytokine signalling cascades and lymphoid cell types and subtypes – it is becoming increasingly evident that not only are NK cells actively involved in controlling HIV infections, but that there are hitherto unsuspected variations among them, and often evidence of specificity in their interaction with infected cells.  Expect to hear much more about these fascinating guys in the future….

There followed a slightly disappointing talk by Shari Gordon, on the use of Human papillomavirus (HPV) pseudovirions (PsV), made in cell culture from co-expression of transfected HPV L1 and L2 capsid proteins and a replicating plasmid vaccine, to immunise mice vaginally.  The idea was to use a mucosa-infecting agent to make a vaccine which induces T-cells and antibody responses at mucosal sites to prevent HIV infection, during the window of opportunity where founder infections are being established.  HPV naturally infects the disrupted vaginal mucosa via interactions of L1 and L2 with receptors on basal keratinocytes – thus it was necessary to disrupt the vaginal epithelium by administration of progesterone and the known inflammatory agent nonoxynol-9 in order to infect.  They used HPV-16 PsV vectoring a SIV gag gene, then boosted with non-cross-reacting HPV-45 PsVs, both expressing red fluorescent protein (RFP) as a marker for in vivo fluorescence tracking.

They got a good response to HPV, and see anti-Gag IgA in vaginal secretions and IgG in serum.  They also see recruitment of T-cells to the site of infection in mucosa – both CD4 and 8 and activated cells.  T-cell responses assayed by intracellular cytokine staining  (ICS) showed that they get CD4+ and 8+ cells in tissue and in blood, which waned over time.  They then set up an experiment to see if systemic priming and mucosal HPV PsV boosting could protect macaques, using a regime consisting of sequential HPV-16, -45 and -58 PsV administration, with and without ALVAC + gag, and gp120 administered with the PsV 45 and 58.  They saw the same gp120 titres at the end of the regimen, with or without ALVAC priming.  They got a Gag-specific response, which expands and recruits T-cells in the genital tract but was lower in blood.  The response was better with ALVAC priming.  There was a primarily monospecific response of both CD4 and 8 T-cells, and primary effector memory.  Upon SIV challenge they saw a similar rate of acquisition despite the immune responses – however, they were only in mid-experiment, and still hoped to see viral control.  She noted the vaccine does not exacerbate the SIV infection rate.

While this was all good science, it was disappointing for a number of reasons.  First, they did not do or report the obvious control, of using DNA only in parallel with PsVs.  Second – in the opinion of my resident HIV/HPV vaccine expert, Anna-Lise Williamson – such vaginal immunisation using PsVs in humans would be a complete non-starter, because it is not ethically acceptable to use agents like nonoxynol-9, which is known to increase HIV infection rates, in a vaccine regimen.  Third, the vaccine did not seem to be very good, despite the supposed advantage of using particles to deliver a DNA vaccine: this is a subject close to my heart, given an interest in both HPV VLPs and DNA vaccines, and I think that oral or intranasal immunisation would have been a far better idea.  Fourth, and although this was not stated, the PsVs are made in immortalised 393TT cells expressing significant amounts of an oncogenic viral protein (polyomavirus T antigen) to enable replication of the vector plasmid – all of which I am sure would be a stern no-no for use in humans.

H Li spoke on the use of recombinant adenovirus vectors in monkeys: he noted that effector memory cells were induced by replicating viruses while non-replicating induced primarily memory cells in blood.  However, people had not looked at mucosal responses.  Accordingly, they used single or double recombinant Ad26 immunisations and showed one could get mucosal T-cells.  With a heterologous Ad5/26 prime/boost they get a potent and widely distributed T-cell response, which they have followed for 4 yrs and still see the responses 2.5 yrs post boost.  Mucosal T-lymphocytes are persistently activated.  They looked at T-cells in PBMC vs colon, duodenum and vaginal tissue: the latter were activated while PBMC were not, so there was only transient activation here.  Memory phenotype shows Tem (effector memory) to Tcm (core memory) evolution in the periphery.  Mucosal T-cells show a persistent Tem1 phenotype.

Ming Zeng revisited the attenuated live SIV vaccine, and its mucosal protective properties.  Live attenuated vaccines offer the best protection yet in monkeys against homologous or heterologous virus challenge – and understanding the correlates would help understand design principles for human vaccines.

They inoculated monkeys with SIV-∆-Nef intravenously, and challenged with repeated intravaginal inoculation.  He showed evidence of a fascinating vaccine-induced Ab concentration at the mucosal border of the monkey cervix, correlated with limited spread and prevention of infection.  They cannot see significant challenge viral growth at portal of entry in vaccinees from 20 weeks post vaccination.  Tissue-associated IgG is concentrated at the port of entry at 20 wk in the cervix and vagina: distribution of the IgG shows one gets plasma cells at the cervix, but also IgG-staining cells especially just underneath the epithelial cell layers.  The cells are epithelial reserve cells and enrich IgG inside cells, presumably by uptake mediated by the neonatal IgG receptor expressed on their surfaces.  This can be shown in vitro by incubating plasma cells with a filter-separated layer of epithelial cells from the female reproductive tract (FRT).

In challenge phase they noticed Ab concentration increased rapidly after challenge in situ.  All genes involved in Ab synthesis were upregulated in challenged monkeys in FRT and germinal centre cells see a dramatic local expansion of plasmablasts after challenge – presumably of memory B cells.

They think the SIV-∆-Nef vaccine converts the FRT to an inductive site for B cell expansion and maturation.  They get 5-10x the amount of IgG produced vs IgA.  They think it is both local recruitment of B cells and activation of local cells that results in the IgG production – which is total and not just HIV-specific IgG.

Again, this is a fascinating result obtained using a controversial vaccine candidate – and one which is not going to go away.

Late afternoon Wednesday was the turn of Symposium Session 02: Recent Advances in B Cell & Protective Antibody Responses – and two talks that took the prize as far as I was concerned were one by Peter Kwong and the following one by Pascal Poignard, both from Scripps in San Diego.

I couldn’t pretend to do justice to the Kwong talk: the graphics were so good, and there was so much detail, that it was like watching a great big complicated shiny machine in motion.  It was very beautiful, but I couldn’t tell you exactly what it is that he did.  Suffice it to say that he introduced us to the concept of mining the “antibodyome” by using structural bioinformatics to get solutions for vaccines by deep sequencing.  A consequence of this was that they could follow the maturation path of specific clones of cells making antibodies binding specific Env epitopes.  An important thing to come out of his talk was a possible reason for why strongly-binding broadly-neutralising antibodies are so rare: they found that, for their preferred target of the CD4 binding site, initially-produced antibodies were of very low affinity and needed a lot of maturation to become strongly neutralising and broadly reactive – which, of course, meant the producing cells were generally selected against and did not make it to being memory B-cells.  Knowing what was possible, however, and being able to make antigens to stimulate those antibodies specifically, would make for a rational vaccine design strategy.

Pascal Poignard described something that has been much in the news lately: the recent discovery of many strongly-binding broadly-neutralising monoclonal antibodies in people living with HIV.  He detailed how the IAVI protocol G search screened 1800 donors, mainly from Africa, for “elite neutralisers”.  They took the top four and did high throughput screening of memory B cells with antigen, and rescued the Ab gene sequences from selected wells, triaged them, and ended up with a selection of potent neutralising MAbs.  These were mostly broadly neutralising, but some were very potent – tenfold better than the previous best.  One group of 5 MAbs – all from the same individual – bind at various sites around the V1 and 2 and 3 loops of Env; another group of 3 from different individuals bind glycans and the V3 loop.  Data suggest protection needs 100x the IC50 value – which was very low for most of them, meaning they could be highly efficacious at low concentration, and synergise each other’s effective in mixtures.  Certain combinations of MAbs would give better protective coverage than others – especially if they did not neutralise the same spectrum of viruses.

Sprawling Bangkok - from the 37th floor

The work raises all sorts of very interesting possibilities, including mimicking the structures bound so well by these MAbs in order to elicit them more frequently, as well as using them therapeutically or in prevention regimes.  As far as antibodies are concerned, it is apparent that we are in a new era of sophistication as regards the potential for both exploiting the natural “antibodyome”, and even designing our own.

There followed a most enjoyable “Faculty Dinner” – my wife got me invited – on the 54th floor of the Centara Grand Hotel, followed by an even more enjoyable sojourn with pleasing beverages on the open deck of the 55th floor, overlooking Bangkok.

Until it rained, anyway.