Sunday, July 20, 2014

Berlin, Boston and now Mississippi


         HIV appears to be a very formidable agent, one that is difficult to deal with in terms of patient care. I have discussed multiple aspects of HIV and focus research on finding a defence is one of the top priority. So when there appears to be an occasional special case of "achieved cure", they make headline and represent an immense interest to science. With the hope they may give us a clue of what the cure can be.

    And this time its a news based on "Mississippi Baby". The child was thought to be a miracle case of complete cure with Anti-retroviral drugs. A case of great hope. And the latest announcement was that of she wasn't cured after all. Am also reminded of another case referred as "Berlin Patient". The case is of a patient who has successfully recovered from a HIV episode after marrow transplant. Oh, yes and then there is this case of Boston Patients, where people appeared to be cured but relapsed. Perhaps these are the most talked about identities in HIV cases other than Gaëtan Dugas, who's on the negative side of Image. Read my earlier post here.

Photo 1: Timothy Brown. Source
      Timothy Ray Brown, more commonly known in science as the "Berlin Patient" is a case of successful battle against HIV. He was diagnosed of HIV and subsequently of acute myeloid leukemia (AML, FAB M4 subtype). His treating haematologist Gero Hütter, decided to transplant bone marrow to treat AML. At the time of presentation, he was in A2 stage of HIV infection. The treating physician, was able to find a rare donor matched for his HLA and has homozygous CCR5-Δ32 allele, known to confer resistance to HIV. He received two stem cell transplants which repopulated his cells knocking the AML off and was resistant to HIV infection. On further long time testing, the patient was negative for detectable viral loads. He has remained so, ever since and is not on ART. Case was published in 2009.

Fig 1: CCR5 structure
       For the first time, There seemed to be fresh new hope. It wouldn't be practical to transplant CCR5-Δ32 homozygous marrow cells to every HIV patient. The estimated allele frequency ranges between 6-16% depending on the study population. Point is that to transplant it should also be HLA matched. Bone marrow itself transplant carries a significant risk and there is ample chances of failing. Clearly everyone cannot be transplanted. But by reverse engineering the exact mechanism we maybe able to find a possible solution to the long standing problem.

      The second story is bone marrow transplantation in 2 patients known as "Boston patients" so called after the Massachusetts city, where they received bone-marrow transplants with cells that were not resistant to HIV. The patients ceased to show viral loads despite being out of drugs for months. The thought was that graft-versus-host disease, an immunological phenomenon seen in transplantation might have destroyed patients cells containing HIV and repopulated by transplant derived cells. However later in a scientific meeting, it was shown that the patients converted back dashing the hopes. The story is well covered here.

     When I think about it, the outcome seems reasonable. The cells donated was not inherently resistant to the HIV invasion unlike in the case of Berlin patient. HIV is known to be shed in low levels. Considering the sudden depletion of T cells, in recipient the HIV levels might be too low to be detectable. Not having received ART, the viral load to be developed will take time. Just as in the case of fresh infection. In this case, the first patient showed relapse after 12 weeks and  the second patient after 32 weeks. It takes just one right virus in the right cell. The report came into light in the end of 2013.

   The child was born prematurely in a Mississippi clinic in 2010 to an HIV-infected mother who did not receive antiretroviral medication during pregnancy and was not diagnosed with HIV infection until the time of delivery. The infant was initiated for active ART therapy, at 30 hours of age. During therapy HIV was detectable in the infant sample. On day 29 the HIV levels were below the detectable levels. The child was on ART for a long time. It was later reported that the child was discontinued ART from 18 months of age. After repeated follow up, by 30 months of age (nearly 2.5 years), routine clinical assays showed that the HIV levels were still undetectable. This represented that the child had controlled the HIV. The study came to be the first successful use of ART for controlling HIV completely even after discontinued use. It also implied that very early ART (within few hours of birth) could probably beat the HIV. The child was known as "Mississippi Baby". The case was published in end of 2013.

    On a routine follow up, at 4 years of age, routine testing showed that HIV levels had been detectable at levels of more than 10,000 RNA copies. Genetic testing has shown that the virus was the same, and doesn't represent a new infection. Read the whole story here. Once again the hopes were thrashed. However, the child remained quiescent for a long time is something that need to be further studied. And perhaps the "complete cure" was declared early.

     At the time of writing, following the news of MH17 flight tragedy (Link), with some leading HIV researchers tragically expired a news of Australian Patients has also come out. In this case too, 2 Australian men treated with stem cell for cancer, have become free from HIV. This time they are on ART and are under constant monitoring. The details were presented in a press conference preceding 20th International AIDS Conference. Once again the results seem interesting. However, as the story has unfolded so far, it is not yet a time to declare "HIV contained". For details and reference go here.

   Perhaps Berlin patient is one of its kind rare case. But learning these cases has given some possible answers and a very challenging question. How much do you consider as a safe time period, to declare a person as "Cured"?
Hütter G, Nowak D, Mossner M, Ganepola S, Müssig A, Allers K, Schneider T, Hofmann J, Kücherer C, Blau O, Blau IW, Hofmann WK, & Thiel E (2009). Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. The New England journal of medicine, 360 (7), 692-8 PMID: 19213682

Yukl SA, Boritz E, Busch M, Bentsen C, Chun TW, Douek D, Eisele E, Haase A, Ho YC, Hütter G, Justement JS, Keating S, Lee TH, Li P, Murray D, Palmer S, Pilcher C, Pillai S, Price RW, Rothenberger M, Schacker T, Siliciano J, Siliciano R, Sinclair E, Strain M, Wong J, Richman D, & Deeks SG (2013). Challenges in detecting HIV persistence during potentially curative interventions: a study of the Berlin patient. PLoS pathogens, 9 (5) PMID: 23671416

Persaud, D., Gay, H., Ziemniak, C., Chen, Y., Piatak, M., Chun, T., Strain, M., Richman, D., & Luzuriaga, K. (2013). Absence of Detectable HIV-1 Viremia after Treatment Cessation in an Infant New England Journal of Medicine, 369 (19), 1828-1835 DOI: 10.1056/NEJMoa1302976

Wednesday, July 16, 2014

Herpes Virus in Neurons- does that benefit?



It has been a long time since I posted some basic virology. There has been a question that I had been asked by a couple of people, and thought it would make a good blog post to answer it. Am sure you have heard of Herpes Virus. Chances are you are infected by at least 2 different Herpes virus and still carry the virus silently without any harm. There are more than 200 different types of herpes virus known, only a handful of the types represents one's which are associated with human infections. There are a variety of Herpes virus in the Earth virome (If I could call it so). Human Herpes virus is classified into α, β and γ sub groups. See Table 1 for details.

So, here's my question. Almost every human is infected with HSV-1 for their lifetime without any significant consequences, unless there is a immuno-compromised condition. Does HSV latency reflect a advantageous position for host? HSV-1 latency is a deeply studied subject. HSV-1 is acquired very early in childhood (less than a year of age). The infection usually resolves by about 2 weeks. The virus then moves axonally (retrograde microtubule-associated transport), reaches the neuronal nucleus and DNA is transported to the nucleus. Once the DNA is in (estimates are that there will be about 10-100 DNA copies in each cell), it circularizes and rests as a extra-chromosomal fraction. Reactivation of virus can lead to retroaxonal transport and establishment of infection.

Fig 1: Events in HSV-1 latency and lytic cycle.
    As per the literature, 3 phases of latency can be recognised- Establishment phase, maintenance phase and reactivation phase. The exact mechanism of how the virus undergoes latency is a question of great research interest. The most common view is that the circular chromosome exhibit a nucleosome like formation, leading to a chromatin like structure. This necessarily implies that factors such as histone modifications are involved in maintenance of latency. This is in good agreement with published results thus far. The only and probably the most important product of HSV detectable inside neurons during latency is the LAT (Latency associated transcript). LAT is a 2 kbp transcript derived by splicing from a less abundant precursor RNA termed minor (m) LAT. The mLAT is transcribed antisense to the ICP0 gene and extends to a polyadenylation signal in the short repeat region. The LAT is a super stable RNA. A summary of events is shown in Fig 1. And how does the Herpes enter activation? Once again, it is not absolutely clear of what is the mechanism. ICP0 was considered as the most likely candidate to date. ICP0, is known to encode a transcriptional activator, which can positively enhance viral mRNA synthesis. More recently VP16 is the favourite candidate.

I have 2 questions to consider here. Taking into account, nearly everyone harbours HSV in their trigeminal neurons, do they represent some sort of benefit to us.

Let us consider the question other way around. If neurons trigger death signals as a form of annihilating the virus inside, the best strategy for virus to be still hanging around is to protect the neurons from destruction. That makes a lot of sense, considering that virus can be sitting down the neuron for decades. For example, In experimental cell lines, it has been shown that CD8 cells are unable to induce apoptosis in latently infected cells. This is thought to be due to LAT’s antiapoptosis activity. This has important implications. For instance, it has been shown in animal models that latency can increase resistance to other infections such as Listeria. I would also think that virus can competitively inhibit other virus trying to gain access into neurons via interference. Such cross protection, might have let us have the trade of benefit.

Second question, Could you some sense consider HSV-1 as a part of neural virome. That is a very tricky question to ask, since the definition of microbiome is not clear. If microbiome is a definition, for microbes that is constituting to be present for a long time in a given niche, then HSV-1 clearly qualifies. It is present for a long-term, and probably beneficial. So why not?
Bigley NJ (2014). Complexity of Interferon-γ Interactions with HSV-1. Frontiers in immunology, 5 PMID: 24567732

Roizman B, & Whitley RJ (2013). An inquiry into the molecular basis of HSV latency and reactivation. Annual review of microbiology, 67, 355-74 PMID: 24024635

Wysocka J, & Herr W (2003). The herpes simplex virus VP16-induced complex: the makings of a regulatory switch. Trends in biochemical sciences, 28 (6), 294-304 PMID: 12826401

Thompson, R., Preston, C., & Sawtell, N. (2009). De Novo Synthesis of VP16 Coordinates the Exit from HSV Latency In Vivo PLoS Pathogens, 5 (3) DOI: 10.1371/journal.ppat.1000352

Jiang X, Chentoufi AA, Hsiang C, Carpenter D, Osorio N, BenMohamed L, Fraser NW, Jones C, & Wechsler SL (2011). The herpes simplex virus type 1 latency-associated transcript can protect neuron-derived C1300 and Neuro2A cells from granzyme B-induced apoptosis and CD8 T-cell killing. Journal of virology, 85 (5), 2325-32 PMID: 21177822

Barton ES, White DW, & Virgin HW (2009). Herpesvirus latency and symbiotic protection from bacterial infection. Viral immunology, 22 (1) PMID: 19210221

Wednesday, July 02, 2014

Ferrets are not humans


Poster 1: Outbreak.
    There is a great deal of controversy in science. But the important problem lies here. People who know the actual science don't talk about it as much as the pseudo science people boast about the non science. This creates a gap, where common people don't understand the complexity of science and the methods to conduct science. So, when there is a talk about a new BSL-4 Laboratory around the town, gain of function research, a concept called as "DURC" pops into the headlines and people are scared the hell out of them. I must acknowledge that some excellent science programmes like the one hosted by Vincent and his team (Link), Radiolab, TED talks and many more, which brings the science to layman language level. However not everyone reads journals and listen to authentic science. Movies like contagion and outbreak have led to assumption in people that someday a lab is going to create a strain that destroys the humanity.. Crap. They are movies, not realities. There is a difference.

   So what does DURC actually mean? DURC stands for Dual Use Research of Concern. The whole concept dates pack to the influenza paper on gain of function experiments (See my previous blog posts here and here). And when some irresponsible reporter posted "Ferret died in experiment", people were not communicated properly by the scientists. Instead opinions were voiced in standard journals which common people never read anyway. Results, too much confusion. And finally the paper was published, showing not a ferret died. Moreover, ferrets are not humans. They are study models. To date, I have never heard of some bio terrorism act using the details in paper. Actually, in practice you can never do it in nature with success.

     Gain of function research, is a controversial hot topic of the day. Media equates the concept with ability to kill. The point is it is not. Let me explain. Let us consider a hypothetical bacteria which causes a particular infection. For understanding molecular mechanisms of how the bacteria works inside the host, scientist in a lab will study the bacteria in a cellular model and (or) animal model. In a normal routine inside the laboratory, genome of the bacteria is played around with, by changing some genes and looking at how it effects the overall outcome of infectious process. That is how virulence is studied. There are well established protocols for these studies, and are done in strict containment facilities. Now let us assume that during generation of the mutant types, a bacteria strain emerged that gained a new function, say more potent toxin than the original it had. In other words, the bacteria has gained a function. But does that pose a threat? The answer is "very unlikely". It is just not potency of virulence that matters in a organism. There is a second, but more important factor called as "Fitness factor".

     You could track a zillion research paper on fitness cost of having a new gene around. For example, MRSA has a additional gene to resist the Methicillin class of antibiotics. But then they also have a reduced fitness of survival. However, in an environment where the organism is constantly exposed to Methicillin, despite the reduced fitness, it helps in survival. Hence the gene is still there. If the MRSA is mixed with a MSSA without the pressure of antibiotics, chances are more that MRSA will vanish from the pool. In other words, MRSA is best adapted to the antibiotic condition. Now let us apply the same logic, to what would happen if there is a gain of function say in influenza. Probabilities are that the influenza is lab adapted. In the outside world, it just wouldn't survive. You may ask, what if by any chance that the gain of function, has lead to both increase in fitness and function? Well, if there is ever such a chance, nature would have already created it, even before you have thought about it. That is the concept of evolution. See my previous posts here and here

      Of course, I by no means say that we should let scientists do anything that they want. That is why there are ethical boards in institutes (Called as IRB, Institutional review Board) which scrutinizes the research and ensures that these work are carried out in strict ethical and standard conditions. Biosafety is a norm of standard research practice. Based on the potential of an organism, in terms of human health, there are different biosafety levels each with stringer conditions for harder pathogens. The highest possible containment system is a BSL-4 Laboratory.

     There is a recent case of resistance against the opening of BSL-4, Boston University’s National Emerging Infectious Diseases Laboratories (NEIDL) laboratory. The laboratory had taken all steps to ensure containment and have passed all the quality checks. However the concern was so high that the matter ended up in court. Recently, the court declared that the facility is ready to go (Link). Here's a funny point. The people who resisted (or lets say protested) doesn't know how a BSL-4 operates. Nor did they care to learn. There is a hour long video that demonstrates everything you need to know about the working of these laboratory. See the YouTube video Threading the NEIDL, Link. I strongly recommend that you watch it.

     A paper was published recently, claiming that laboratories represent a area of potential pandemic (Link). I have no clue why an opinion in biological safety is published in a magazine of Atomic science. Anyway, TWiV 287 (Link), has a very detailed discussion of how the paper is totally flawed and doesn't represent anything scientific. I cannot agree better.

       And now there is a new controversy, just published in Nature news that inspired me to write this page. The study in question is led by Kawaoka, to investigate what made the 1918 H1N1 virus hyper virulent. As per the report, there is an apparent disagreement between University of Wisconsin–Madison and US National Institute of Allergy and Infectious Diseases (NIAID). I have Quoted the article below (in blue, with my comments inserted in red).

The team used genes from wild avian H1N1 viruses that coded for proteins resembling those of the 1918 virus to construct new viruses (Gain of function experiment). The new viruses were not only able to spread between ferrets — the best current model (It is a model. Ferrets are not humans) for human flu transmission — they were also more virulent than the original avian viruses. The team therefore argues that there is a high risk that a 1918-like virus could emerge naturally (And what crap conclusion is that??)

Photo 1: Ferret
   I must address a question here so as to clarify. First, science works on something called Risk- Benefit ratio. When an experiment such as mentioned above is conducted, the aim is to determine what are the factors accounting for transmission. To avoid the chances that the virus is a real threat, the chances of it getting into humans is avoided by using a model animal (That is why we use models after all and not humans). Ferret represents the closest model in terms of if it can be infected by the influenza strain under study. But mind you, ferrets are closest, not humans. We differ significantly. Somebody argued that if ferrets are not humans then why do the experiment at all. The answer is, it provides us with a road map of the possible dynamics which though doesn't match exactly still keeps us prepared. As an example, soldiers are first taught to fight the dummy enemies, not the real ones, though there is a huge difference between dummy and real. The benefit is very clear and high. In contrast, what is the risk? Virtually zero. There is containment and it is infinitesimally improbable that a ferret will sneeze into human face and the so transferred virus is human adapted enough to cause an infection.

Let me throw a question around. Is it that there never has been an accident in laboratory leading to problems? The answer is clearly a no. So how is it justified? Let us consider a fresh report again from this week in nature. CDC has reported that it had supplied anthrax to 2 other BSL-2 laboratories. Live bacteria for non published reasons survived the inactivation step, and were not detected before samples were sent out. If you had consider from the same report that that has been 727 incidents of theft, loss or release of Select Agents and Toxins in the United States between 2004 and 2010, resulting in 11 laboratory-acquired infections and no secondary transmission, that accounts to about 1.51% risk ratio. Consider, routine clinical laboratory practice and there is roughly similar risk ratio. Further, at least in this case, there is immediate remedial measures taken to quarantine the problem.

To conclude let me say that humans as a species has evolved to fear for small dangers in comparison to the vice versa. So when somebody puts an headline and says "Lab creates pathogenic strain", it is more feared and attacked than when someone says "Cure found". And media knows how to cash in the headlines, which potentially misleads people.

Fauci AS, & Collins FS (2012). Benefits and risks of influenza research: lessons learned. Science, 336 (6088), 1522-3 PMID: 22723407

Declan Butler& Brendan Maher (2014). Risks of flu work underrated. Nature News, 2014: 10.1038/511013a

Biosafety in the balance Nature, Nature News 2014. 510 (7506), 443-443 DOI: 10.1038/510443a