Wednesday, October 18, 2017

Usutu virus

At least according to me flavivirus members are one of the most difficult to understand classification. Thats because, they have so many different types of viruses by their characters and too many different types. Almost every virus in this group has at least a part of its life cycle in an animal. Once in a while they pop up in a limited geographical region and are noticed by the global infection research community.

Photo 1: Eurasian blackbird (Turdus merula).
Source
One such virus which has recently grabbed some attention is Usutu virus. Usutu virus (USUV) is a member of flavivirus closely resembling Japanese encephalitis complex. It is a Group IV member, with + single stranded RNA genome. It was first identified in South Africa in 1959. USUV first came into significant notice in 2001 when there was a massive black bird mortality in eastern Vienna, Austria. Subsequently, this virus has been identified in several geographical locations. But until now, USUV was thought to be limited to bird population, except for 4 confirmed cases in humans ever to be reported. In a surveillance study, in Austria, 54 of 208 individuals tested positive for USUV-neutralizing antibodies, thus indicating exposure.
Fig 1: Phylogenetic analysis of several members of the 
JEV group and selected other mosquito-borne flaviviruses.
As a part of blood screening program in Austria, samples from July to August 2017, 12047 blood samples from donors in eastern Austria was screened for West Nile virus (WNV) infection. Of these 7 samples were found to be positive for WNV by nucleic acid testing. WNV positive samples were follow up tested by WNV- and Usutu virus (USUV)-specific RT- and RT-qPCR assays for confirmation, which showed that of the 7 positive, 6 were USUV positive only one was WNV positive. This was further confirmed using sequence analysis. Interestingly, none of these donors had or later developed any clinical symptoms.

Norbert Nowotny from Austria who is an author in the most recent paper on USUV comments, "Flavivirus-positive blood donations, both West Nile and Usutu virus positives are discarded and consequently do not pose any risk to recipients of blood donations. However, there are a number of European countries, in which West Nile virus infections did not yet occur but in which Usutu virus circulates. In these countries blood donations may not be screened for flaviviruses. On the other hand, blood recipients are frequently immunocompromised persons, in which an Usutu virus infection may result in severe disease. To increase awareness of this possibility was one of the main goals of our second study".

Considering that USUV seropositivity and genetic evidence of virus in healthy blood samples are found without any associated clinical symptoms it currently appears unlikely that this virus is significantly causing a human infection. But, given its close genetic relatedness to JEV and WNV, the virus can make a jump anytime, which is proposed to be monitored.

Reference:

Bakonyi T, Jungbauer C, Aberle S, Kolodziejek J, Dimmel K, Stiasny K et al. Usutu virus infections among blood donors, Austria, July and August 2017-Raising awareness for diagnostic challenges. Eurosurveillance. 2017;22(41).

Ashraf U, Ye J, Ruan X, Wan S, Zhu B, Cao S. Usutu Virus: An Emerging Flavivirus in Europe. Viruses. 2015;7(1):219-238.

Bakonyi T, Erdélyi K, Brunthaler R, Dán Á, Weissenböck H, Nowotny N. Usutu virus, Austria and Hungary, 2010-2016. Emerg Microbes Infect. 2017 Oct 11;6(10):e85. doi: 10.1038/emi.2017.72.

Thursday, October 05, 2017

Nobel Awards- 2017

Nobel Prize is considered as the most prestigious award for research accademics. The recipients of the award are chosen by the Nobel foundation constituted by Nobel committee of Royal Swedish Academy of Sciences, Nobel committee of Karolinska Institutet and Norwegian Nobel Committee. The award consists of a citation, gold medal and money. More than the award amount, the fame is considered as far superior.

1. Physiology / Medicine:

Photo 1: Michael Rosbash (left), Jeffrey Hall (centre)
and Michael Young (right). Source
The award goes to three scientists- Jeffrey Hall and Michael Rosbash (from Brandeis University Waltham, Massachusetts) and Michael Young (Rockefeller University) for their studies on circadian clocks. Circardian rhythm in simplest terms is a endogenous, entrainable biological system which oscillates over a period of cycling time aproximately 24hrs. It is widely seen across species from cyanobacteria to humans. The field of study called as "Chronobiology" has been shown to have important affects including human health. The functioning of circardain rhythm has been  shown to be strongly affected in conditions such as sleep disorders and several mental health disorders such as Schizophrneia.

2. Physics:

Photo 2: Rainer Weiss (left), Barry Barish (centre), and
Kip Thorne (right). Source
This award has been announced to three scientists- Rainer Weiss (Massachusetts Institute of Technology, Cambridge), Barry Barish and Kip Thorne (California Institute of Technology, Pasadena) for their work on detection of gravitational waves at Laser Interferometer Gravitational Wave Observatory (LIGO). Gravitational waves are ripples in the curvature of space-time that are generated in certain gravitational interactions and propagate as waves outward from their source at the speed of light. This phenomenon was predicted in 1916 by Albert Einstein on the basis of his theory of general relativity. Basically, when 2 massive objects such as black holes collide with each other they distort the space time around which travels through space. The pattern of this wave helps understand several astrophysical phenomenon.

3. Chemistry:

Photo 3: Jacques Dubochet (left), Joachim Frank (centre)
and Richard Henderson (right). Source
The award has been given to Richard Henderson (MRC Laboratory of Molecular Biology, Cambridge) Joachim Frank (Colombia university), and Jacques Dubochet (University of Lausanne, Switzerland) helped to develop cryo electron microscopy. Cryo-electron microscopy  or famously referred to as cryo-EM is a type of electron microscopy where the sample is studied at highly frozen temperatures. Cryogenic processing gets around the problem of treating the cells or molecules with several reagents, instead it could be seen in its native form. Cryo EM is now commonly used to study structure of viruses and there are extended 3D versions of the technique. Cryo EM- method is also slowly replacing X ray crytsallography method to study biomolecule structure.

Apart from the above Nobel Prize in Literature, Nobel Peace Prize and Sveriges Riksbank Prize in Economic Sciences in Memory of Alfred Nobel are given, for which the winners are yet to be announced as of at the time of writing this post.

Saturday, September 30, 2017

Which viruses maybe seen in human semen

One of the least understood questions in virology in a recent turn of outbreaks like Ebola and Zika are that there are reports of these viruses being transmitted sexually. Now that we know that semen by itself possibly harbours a microbiome (Link) there definitely is a good chance that many different microbes can harbour in reproductive tissues such as testis. But there is no comprehensive literature on all possible viruses infecting the male reproductive tract. 

A meta-analysis which searched for all the literature available on evidence of virus presence in semen turned up 3,818 PubMed search results. Screening all the literature, the authors concluded that 26 viruses can survive in human semen which can seed a viremia. A summary is shown in Table 1.
Table 1: Viruses that are capable of causing viremia and found in human semen. Source
It should be noted that not all viruses listed have a defined capability to be sexually transmitted. But their biological effects on variables such as sexual health is not known. It is also known what is the duration of residence and how far they can seed viremia, serve as latency and what is the likely concentration. As Alex Salam puts it, "Clinicians need to consider the possibility that traditionally non-sexually transmitted viruses can persist in semen, and this, therefore, raises the possibility of sexual transmission. Detection means that evidence of viral genetic material or viral protein was found in semen. It’s important to note that this does not mean that the virus is viable, i.e., capable of replicating. To prove this, the virus needs to be isolated and grown in cells or animals. For many of the viruses, this test has not been done, so we don’t know whether virus is viable or not."

This paper needs to be followed up with laboratory tests to establish the results. Further, this list is definitely not complete. But at least we have a more likely and possible catalogue of which viruses may be found in semen.

References:

Salam AP, Horby PW. The breadth of viruses in human semen. Emerg Infect Dis. 2017. https://doi.org/10.3201/eid2311.171049

Friday, September 29, 2017

Zika Virus- Update III

Photo 1: Digitally-colorized TEM of
Zika virus. Source
The last I wrote about Zika was almost more than an year ago (Link). Ever since, literature on Zika Virus (ZIKV) has grown tremendously. It is too much to ask for a summary of everything that is known about zika to date. For all my posts on Zika, refer this link. In this post I will give the most recent updates of interest.

Zika virus has roughly a global distribution and 79 countries are in the spotlight. The geographical regions affected are categorised into 4 categories (Category 1-4). Category 1 represent countries with new introduction of Zika virus since 2015 with ongoing transmission. Category 2 are areas with either the evidence of virus circulation before 2015 or with ongoing transmission of the virus that is no longer in the new or re-introduction phase, but where there is no evidence of interruption. Category 3 are regions with interrupted transmission and with potential for future transmission. Category 4, where the Aedes aegypti mosquito that spreads the disease exists, but there has been no reported transmission of the virus.
Fig 1: Global map of ZIKV infection categorised by country. Source
There are 2 hypothesis on why the ZIKV infection has emerged. The first is that the ZIKV infection was indeed causing infection earlier. But there was no targeted testing to detect them. The other is genetic hypothesis. As per this view, genetic change might have resulted in emergence of a virus strain with greater epidemic potential and virulence, causing epidemics with more severe disease. In a just published study a team of researchers have explored this hypothesis. The team strated with analysis of genetic differences between modern Zika strain and an ancestral strain isolated from a patient in Cambodia in 2010. Based on the initial analysis, a single serine to asparagine substitution (S139N) in the viral polyprotein was predicted as important. This was then proven with a mouse model which showed that virus with the S139N mutation caused the most damage to neuronal cells. As the author of the paper Chen-Feng Qin comments, "Besides host factors, such as low immunity to the virus in affected communities, there are definitely some other unknown viral proteins or amino acids that may contribute to the complex pathogenesis of microcephaly—independently or synergistically. Our study identified a unique genetic determinant that links to severe microcephaly".

The exact mechanism of pathogen invasion is not known. There is some indication that the virus targets neuronal progenitors in the developing brain. Research has suggested that AXL receptor tyrosine kinase is the cellular receptor for the virus. But most probably this is not the only receptor. Given that the ZIKV infection targets glial cells, researchers have tried to use Zika strain to attack glioblastoma in a laboratory model.

Photo 2: PET  brain images post-infection
with Zika virus. Source
In a recent research using PET scanning (using a probe [18F] DPA-714) scientists have imaged the brain inflammation following Zika virus infection in mice. They found that levels of Zika virus in the mouse brain increased from day 3 to day 10 post-infection. During the period, the mice showed a 2- to 6-fold increase in global brain neuroinflammation. The study highlited that despite Zika affecting local brain tissue parts, the inflammation extends to all parts of the brain.

The focus of research currently is the cross reactivity between Zika and Dengue. Zika and Dengue are closely related viruses. Both the viruses show antibody enhanced infection and hence there is the question as to how does dengue vaccine affect Zika infection. There is no clear answer to this question. There is some opinion that dengue infection enhances zika infection. More recently, it has been suggested that antibodies against DENV E-dimer epitope (EDE) not only neutralizes the dengue virus in mice, but also protects both adults and fetuses from Zika infection by neutralisation. The different findings in various papers is probably based on the various types of targetting antibody that is tested.

Zika has gained strong interntational attention and there is a great focus on studying the Zika transmission, pathogenesis, treatment and vaccine.

References:

Yuan etal. A single mutation in the prM protein of Zika virus contributes to fetal microcephaly. Science (2017). DOI: 10.1126/science.aam7120

David Baud, Duane J Gubler, Bruno Schaub, Marion C Lanteri, Didier Musso. An update on Zika virus infection. Lancet (2017). http://dx.doi.org/10.1016/S0140-6736(17)31450-2

Estefania Fernandez, Wanwisa Dejnirattisai, Bin Cao, Suzanne M Scheaffer, Piyada Supasa, Wiyada Wongwiwat, Prabagaran Esakky, Andrea Drury, Juthathip Mongkolsapaya, Kelle H Moley, Indira U Mysorekar, Gavin R Screaton, Michael S Diamond. Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection. Nature Immunology, 2017; DOI: 10.1038/ni.3849.

Kyle Kuszpit, Bradley S. Hollidge, Xiankun Zeng, Robert G. Stafford, Sharon Daye, Xiang Zhang, Falguni Basuli, Joseph W. Golden, Rolf E. Swenson, Darci R. Smith, Thomas M. Bocan. [18F]DPA-714 PET Imaging Reveals Global Neuroinflammation in Zika Virus-Infected Mice. Molecular Imaging and Biology, 2017; DOI: 10.1007/s11307-017-1118-2

Sunday, September 17, 2017

Compound isolated from C difficile act against C difficile: Avidocin-CDs


Clostridium difficile represents a unique problem in health care system. C difficile is not affected by a wide range of antibiotics and they start showing up when gut microbiome is depleted due to antibiotics. C difficile is often treated with antibiotics such as vancomycin, metronidazole and fidaxomicin which cause further disruption of the resident microbiota leading thus increasing chances of relapse. A faecal microbiome transplant (FTM) is an option but it comes with some challenges. For example, It is nearly impossible to rule out every possible infection from a healthy donor and we are technologically incompetent yet to find the right best combination of microbiome for the gut. The best answer would be an antibiotic that kills only and only C diff. That means we have to be extremely specific.

Fig 1: Electron micrograph of negatively stained
purified diffocin particles isolated from CD4 strain.
C difficile is a highly competitive bacteria. C difficile strains compete with each other and they produce an R-type bacteriocin (High-molecular-weight or phage tail-like) which is called as diffocins. In a generalised sense, R-type bacteriocins attack target cells by specific receptor-binding on the surface. This is followed by sheath contraction and insertion of the core through the envelope of the target bacterium. The diffocins are ultra specific in their activity and their receptor binding activity can be modified to attack C difficile in general by modifying its binding to specific receptors. 

Table 1: Sensitivity of various bacteria to modified diffocins.
Source
Table 1 shows a summary of the sensitivity of various bacterial strains to heterologously expressed, recombinant diffocins. A company that is currently working on using these diffocins and their modified version- Avidbiotics Corp (California based Biotech company) have named them as Avidocin-CDs. One of such constructs Av-CD291.2 had been found to have wide spectrum activity against a range of hypervirulent C difficile strains tested, without affecting the microbiome.

There is sufficient literature evidence to indicate that avidocins are highly specific for C difficile and able to survive transit through mouse GI tract.

In the latest paper, the story becomes better now. The team of scientists from the University of Sheffield, AvidBiotics Corp, and the University of Glasgow have published a study on Avidocin-CD291.2 on how it could be extended for clinical use. Analysis of rare Av-CD291.2–resistant mutants enabled identification of S-layer protein A (SlpA) as the target. The paper showed that Av-CD291.2–resistant mutants lack an S-layer. The lack of S-layer also introduces a high sensitivity to innate immune molecules combined with sporulation defects. These S-layer mutant strains survived poorly in the standard charcoal medium which is used to transport C difficile strains. Interestingly, acquisition of Avidocin-CD resistance results in loss of toxin production and complete loss of virulence.

As Dr Robert P. Fagan, senior corresponding author comments, "We discovered that the weapons naturally produced by C difficile and those engineered by our colleagues at AvidBiotics were using certain proteins in the S-layer to identify which strains to target. The C difficile S-layer is unique to these bacteria, which explains why Avidocin-CD killing is so specific. Scientists at AvidBiotics Corp were then able to engineer different versions of Avidocin-CD to target 12 of the 14 known types of S-layer."

Reference:

Joseph A. Kirk, Dana Gebhart, Anthony M. Buckley, Stephen Lok, Dean Scholl, Gillian R. Douce, Gregory R. Govoni, Robert P. Fagan. New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability. Science Translational Medicine, 2017; 9 (406): eaah6813 DOI: 10.1126/scitranslmed.aah6813

Tuesday, September 05, 2017

Hypervirulent Klebsiella pneumoniae

One of the most common ideas associated with clinical antibiotic resistance is the fitness factor. I have in multiple posts talking about this idea in my previous posts. The idea is that when a bacteria acquired genes for antibiotic resistance there is a fitness cost associated with it. Until and unless there is this constant antibiotic threat to the bacteria, it is not so useful for the bacteria to keep resistance genes, since there is a cost associated with maintaining that gene. There are several experiments that showed at least for some drugs that if you mix a sensitive and resistant phenotype of a given organism and allow it grow together on an antibiotic free condition, the sensitive strain tends to dominate easily.

The idea is all fine and good in laboratory conditions. In a series of 5 hospital acquired pneumoniae infection (All fatal) following surgery in a Chinese hospital, a Klebsiella pneuomoniae ST11 strain has been isolated which is not only an MDR (Multi drug resistant) but also a hypervirulent strain.

The study, in summary, basically went something like this. There were 5 back to back pneumoniae cases following surgery for traumatic cases who developed pneumoniae and died of it. The first one in the series was identified as the most probable patient zero (Index case). The other four patients were located in different wards with overlapping stays. In all the cases multiple samples were taken and all the Klebsiella pneumoniae strains isolated were fully characterised using phenotypic and genetic approaches. In total 21 non-repeated carbapenem-resistant K pneumoniae strains were recovered from various clinical specimens of the five patients

What was really interesting to me was that they did a string test, human neutrophil assay and a wax moth virulence test to establish its hypervirulence status. These tests are really valuable models and so I think I should explain these in a little bit of detail.

Photo 1: Positive “String test” on a hypervirulent
strain of K. pneumoniae. Source
String test (Don't confuse this with the string test done to identify Vibrio cholerae), is a kind of qualitative marker used to test the hypermucoviscous phenotype seen in Klebsiella pneumoniae which are an indicator or hypervirulence. Basically, you grow the organism on a 5% sheep blood agar at 37°C overnight. The string test is positive when a bacteriology inoculation loop or needle is able to generate a viscous string > 5 mm in length by stretching bacterial colonies on an agar plate. See Photo 1. In this paper, authors reported that the string test was positive for all five strains, each producing strings longer than 20 mm.

Fig 2: Neutrophil Survival of K pneumoniae strains.
Source
Neutrophil assay is a test to see how good the bacteria resist killing by the neutrophil in laboratory conditions. In principle,  neutrophils obtained from healthy volunteers. A well containing 10⁶ neutrophils and 10⁶ CFU of opsonised K pneumoniae in RPMI/H medium is prepared at 37°C. A small sample is taken at intervals and bacteria is plated on Luria broth agar. Survival is calculated as a percentage of CFUs with reference to controls. For the study, K pneumoniae 4 and 5 (representative strains), two classic ST11 strains FJ8 and FJ9 (known to be not hypervirulent strains) and two known K1 hypervirulent K pneumoniae strains 1088 and 91 along with PC K pneumoniae which was removed for its virulence plasmid were studied. Fig 1 shows the results. You can clearly see that the strains 4 and 5 were really evading the neutrophil killing.

Fig 2: Virulence potential of isolated strains.
Source
Another test for virulence was done using Galleria mellonella or honeycomb moth. Basically, the bacteria are incubated with the larvae and you look for the ability of the larva to survive. For this study, cultures of K pneumoniae strains were washed with PBS and larva was infected with the bacteria and survival rate of the larvae was studied. See Fig 2. 

The study does provide a compelling evidence that the strains they isolated were hypervirulent. Yes, they were also MDR strains but most of the K pneumoniae strains isolated globally are MDRs. But mind you they are not super bugs. I wasn't sure if these strains were resistant to drugs like Colistin and from the data they appear tigecycline sensitive.

I take this study as a proof that ST11 type which is a common circulating type in Asia is capable of hypervirulence. They have a 170 kbp plasmid (pVir- CR-HvKP4) which makes it hypervirulent, multidrug resistant, and transmissible. Considering so many people are dying from Klebsiella pneumoniae infections it may be worthwhile to test how many percentages of it is actually hypervirulent.

Reference

Danxia Gu, Ning Dong, Zhiwei Zheng, Di Lin, Man Huang, Lihua Wang, Edward Wai-Chi Chan, Lingbin Shu, Jiang Yu, Rong Zhang, Sheng Chen. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. The Lancet Infectious Diseases, 2017. https://doi.org/10.1016/S1473-3099(17)30489-9

Thursday, August 17, 2017

Gonococcus superbug: Current Status

Photo 1: Neisseria gonorrhoeae.
Neisseria gonorrhoeae or what is commonly known as the gonococcus is sounding serious alarms all over the globe. In a post almost an year ago, I talked about how gonococcus is slowly rising to the status of true superbug. Gonococcus is responsible for a sexually transmitted disease called as Gonorrhea. Decades ago, this was absolutely treatable with a simple penicillin. However, they have now acquired several genes that makes it more untreatable.

Table 1: Resistance pattern of Gonococcus. Source
Here is the summary of current issue based on WHO reports in June. WHO has estimated that nearly 78 million people are infected with gonococcus. There are countries that are reporting their annual statistics and some countries that dont. The current recommended regimen for gonorrhea treatment is a combination of azithromycin and ceftriaxone. Table 1 shows a summary of number of countries in different WHO regions reporting gonococcal isolates with resistance to azithromycin and ciprofloxacin, and decreased susceptibility or resistance to extended-spectrum cephalosporin (Cefixime and/or ceftriaxone) for at least 1 year from 2009 to 2014. It should be noted that the report is based on data from 77 countries. Most African nations where the percentage of gonorrhea is expected to be higher has not reported. Interestingly, WHO has reported 3 confirmed cases of gonococcus that are resistant to all the anitbiotics tested. 

“These are cases that can infect others. It can be transmitted. And these cases may just be the tip of the iceberg, since systems to diagnose and report untreatable infections are lacking in lower-income countries where gonorrhea is a ctually more common.”
-Teodora Wi (Department of Reproductive Health and Research, WHO)

“To control gonorrhoea, we need new tools and systems for better prevention, treatment, earlier diagnosis, and more complete tracking and reporting of new infections, antibiotic use, resistance and treatment failures. Specifically, we need new antibiotics, as well as rapid, accurate, point-of-care diagnostic tests – ideally, ones that can predict which antibiotics will work on that particular infection – and longer term, a vaccine to prevent gonorrhoea.”
-Marc Sprenger, (Director of antimicrobial resistance, WHO)

By now you understand that gonorrhea is a serious issue and needs to be addressed qucikly. Vaccine is an excellent choice. But vaccine research in gonorrhea isn't so great and there are no candidates in sight that are ready to be launched. Howewer, there is a silver lining.

A study was recently published which retrospectivly looked into vaccine effectiveness of outer membrane vesicle meningococcal B vaccine (MeNZB) in a case-control study of patients at sexual health clinics aged 15–30 years. They found that the gonorrhea rate among teens and young adults who had received a meningitis B vaccine during an emergency campaign in the early 2000s was significantly lower than the rate seen in people of the same age who weren’t vaccinated. The estimated vaccine effectiveness of MeNZB against gonorrhoea was about 31%. Of course its a chance observation and some form of clinical trial needs to be done to have more credibitily for the claim and get a more realistic estimate. There are also similar reports of decline in gonorrhea rates follwing meningococcal vaccine in Cuba, and Norway.

There is a lot of effort currently in trying to come up with new antibiotics especially ones that can target such superbugs. A compound referred to as closthioamide has been shown to have promising results against drug resistant gonococcus.

Fig 1: Structure of Clostioamide.
Source
Closthioamide (CTA) was first discovered and isolated from Anaerobic Bacterium Clostridium cellulolyticum in 2010 which was initially tested as a possible compound against multi drug resistant Staphylococcus. It functions through attacking DNA gyrase. In brief, the researchers tested 149 strains isolated from patients, 8 WHO reference strains of of N. gonorrhoeae and 4 commensal Neisseria strains were tested. CTA performed really well against 146/149 (98%) of clinical gonococcal strains at ≤0.125mg/L. The study also noted that two N. perflava strains had the highest CTA MIC (>1 mg/L).

As the senior author of the study John Heap comments, "The imminent threat of untreatable antibiotic-resistant infectious diseases, including gonorrhoea, is a global problem, for which we urgently need new antibiotics. This new finding might help us take the lead in the arms race against antimicrobial resistance. We believe there are many undiscovered antibiotics out there in nature, but they are difficult to find and test. For example, the bacteria which produce closthioamide naturally make only tiny amounts that are not enough to test or use, so we had to chemically manufacture it ourselves by mimicking its natural structure. The next step will be to continue lab research to further assess the drug's safety and effectiveness. Despite showing tremendous promise, it will be a number of years before, and if, we can use the drug in real life human cases."

As of now there is no clear answer as to how to tackle the globally spreading true superbug "gonococci" is to be controlled. Perhaps the best method is the same as standard STD prevention methods.

References:

1. Wi T, Lahra M, Ndowa F, Bala M, Dillon J, Ramon-Pardo P et al. Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for international collaborative action. PLOS Medicine. 2017;14(7):e1002344. 

2. Petousis-Harris H, Paynter J, Morgan J, Saxton P, McArdle B, Goodyear-Smith F et al. Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study. The Lancet. 2017; doi: 10.1016/S0140-6736(17)31449-6. [Epub ahead of print]

3. Miari V, Solanki P, Hleba Y, Stabler R, Heap J. In vitro susceptibility to closthioamide among clinical and reference strains of Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy. 2017;:AAC.00929-17.

4. Lincke T, Behnken S, Ishida K, Roth M, Hertweck C. Closthioamide: An Unprecedented Polythioamide Antibiotic from the Strictly Anaerobic Bacterium Clostridium cellulolyticum. Angewandte Chemie. 2010;122(11):2055-2057.

Wednesday, August 09, 2017

Scientists find a quick method to get Monoclonal Antibodies of interest.

A lot of new emerging infectious diseases are no on global radar and that highlights how unprepared we are in fighting it. Most of these are geographically limited and terminate with a few countable number of cases. The one's like Influenza variants, Zika and Ebola have been more rampant. Though a short term solution is to administer antibiotics or quarantine, the best approach is to vaccinate. The centre of this whole problem lies in B cells.

Fig 1: B cell activation. Source: Kuby Textbook; 5e
I need to visit back the B cell activation pathway. B cells are a type of lymphocytes that is involved in making antibodies. B cells by nature are inactive and have to be activated specifically. The individual lineage of B cells can make antibodies against a specific antigen. These inactive B cells which are competent enough to start making antibodies, provided they have the right signal, is called as immunocompetent B cells.

Depending on the nature of the antigen, there are two modes of B-cell activation. TH cells dependent (TD) and TH cell independent (TI). There are 2 types of signals that are required as membrane events, to activate a B cell. The first activation signal is an antigen binding to B cell receptors (BCRs). Once bound, the antigen is internalized by receptor-mediated endocytosis, digested, and complexed with MHC II molecules on the B cell surface. The second activation signal (also referred as the costimulatory signal) is CD40/CD40L interaction. See Fig 1. Once activated they go on to mature and convert to B cells which start making antibodies. There is some evidence that this is not completely true and there are more signals in the interaction. For example, TLR is possibly the third signal. 

There have been several previous attempts in the laboratory to replicate this process in the laboratory condition. Previous studies have shown that patient-derived B cells when treated with CpG oligonucleotides, they stimulate every B cell in the population. CpG oligonucleotides are short single-stranded synthetic DNA molecules that contain a cytosine triphosphate deoxynucleotide followed by a guanine triphosphate deoxynucleotide. The CpG is mainly recognised by TLR 9, which is expressed in B cells and Plasmacytoid dendritic cells and is thus an excellent immunostimulant. Antigen-dependent activation of B cells in-vitro is difficult to achieve result because the wide haplotype variation of MHC IIs necessitates the use of unique T cells specific to a particular MHC II to activate B cells in vitro.  This problem was solved by the team led by Facundo Batista, from the Francis Crick Institute in London based on which the current paper is built.

The researchers started with coated streptavidin polystyrene nanoparticles containing a mixture of biotinylated anti-κ antibody and the TLR ligand CpG. The team showed that by treating the patient derived B cells with the coated nanoparticles and the appropriate antigen. In short, CpG oligonucleotides are only internalized into those B cells that recognize the specific antigen coated, and these cells are therefore the only ones in which TLR9 is activated to induce their proliferation and development into antibody-secreting plasma cells. 

The team has shown that the results are replicable when done with different bacterial and viral antigens (such as tetanus toxoid and proteins from several strains of influenza A, HIV gp120). The studied showed specifically that in vitro stimulation of memory B cells with particulate antigen-CpG selectively enriched the frequency of CD27hi/CD38hi antigen-specific plasma cells irrespective of the nature of the antigen that was chosen. So technically in proof, this method can be applied to any antigen. Further, it was achieved in a very short time, almost a week.

This novel method is much superior to several other methods such as phage display, EBV immortalization, yeast display, and humanized animal models since it doesn't rely on a large scale screening and identification. Basically, this method allows for selective stimulation of memory B cells from healthy donors, leading to proliferation and differentiation into plasma cells that produce antigen-specific antibodies, even in antigen-naive donors (They demonstrated by showing you could develop antibodies against HIV from cells derived from HIV negative donors), which means making therapeutic vaccines (Antibodies) could be very fast.

As Facundo Batista explains, "Specifically, it should allow the production of these antibodies within a shorter time frame in vitro and without the need for vaccination or blood/serum donation from recently infected or vaccinated individuals. In addition, our method offers the potential to accelerate the development of new vaccines by allowing the efficient evaluation of candidate target antigens."

References:

Irene Sanjuan Nandin, Carol Fong, Cecilia Deantonio, Juan A. Torreno-Pina,Simone Pecetta, Paula Maldonado, Francesca Gasparrini, Jose Ordovas-Montanes, Samuel W. Kazer, Svend Kjaer, Daryl W. Borley, Usha Nair, Julia A. Coleman, Daniel Lingwood, Alex K. Shalek Eric Meffre, Pascal Poignard, Dennis R. Burton, and Facundo D. Batista. Novel in vitro booster vaccination to rapidly generate antigen-specific human monoclonal antibodies. The Journal of Experimental Medicine, 2017 DOI: 10.1084/jem.20170633.

Eckl-Dorna J, Batista F. BCR-mediated uptake of an antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell formation. Blood. 2009;113(17):3969-3977.

Tuesday, July 25, 2017

Dopamine says "Make antibodies"

Photo 1: Lymphatic system
in brain. Source
For a long number of years nervous system and immunological system have been seen as two separate systems. In the last couple of decades this idea has been strongly questioned. Central nervous system which has been assumed to be devoid of immune activity is now known to harbour immune system of its own (Link). Their location was quite close to prominent blood vessels. There has been some proof that neural system could in part regulate neural activity also (Link). In 2015, Louveau et al reported that the brain has a lymphatic system of its own. The anatomical discovery was surprising since the vessels’ were hidden in a location deep within the brain. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), is a marker of lymphatic system. LYVE1 immunostaining of whole-mount meninges is shown in Photo 1 showing distribution of brain lymphatics.

There are several papers that have established that neuroimmune interactions are 2 way. Classical nervous system molecules such as dopmaine can act on immune cells. For example, T cells express several dopamine receptors (DARs). It has been established that stimulation of specific DARs on Dendritic cells and T cells, influence CD4+ T cell differentiation into Th1 or Th17 inflammatory cells. Dopamine receptors are universally expressed in T cells, dendritic cells (DCs), B cells, NK cells, neutrophils, eosinophils, and monocytes. Fig 1, shows an example of established pathways on how neurotransmitters affect T cell response. Ofcourse, the reverse is also true, though the phenomenon is less well studied. A really good example is cytokines. IL-6 is now highly implicated as a Neuropoetin (Stimulate Neuronal growth), though mechanism is not clearly defined.

Fig 1: Neurotransmitter-mediated regulation of T cell response. Source
So far, so interesting. There is convincing evidence that dopamine receptor is important. But having a receptor is one thing. There has been some research earlier suggesting that the immune cells can themselves also make dopamine and it has not been clear as to what is the role. And thats the new story.

Making an antibody is a very tightly regulated process. When the immune system encounters a foriegn molecules the T cells delivers signal to B cells in a complicated molecular process. In germinal centres, highly mobile T cells and B cells specific for the same pathogen can directly interact with each other through the formation of dynamic specialized surface structures called T–B immunological synapses. In a new study by Papa etal, showed that the Follicular helper T cells or TFH (They are also known as Follicular B helper T cells) use dopamine as cargo loader for immune molecules which mediates a T-B synapse which triggers B cell maturation.

Fig 2: Graphic model of the proposed positive feedback
between human TFH and germinal centre B cells.
Source
The first experiment goes on to show that Chromogranin B (CGB) is present in human tonsils, spleens, and lymph nodes and determined that the T cells isolated from these samples icontained granules filled with dopamine. Using a method called live-cell RNA detection CGB was shown to be present in high quantities of human germinal centre TFH cells but not so much in other T cells. Subsequent experiments showed that dopamine was not really there in detectable amounts in cells other than TFH cells. Next set of experiments showed that human TFH cells released dopamine on stimulation by germinal-centre B cells and it upregulated the ICOSL on the cell surface of germinal-centre B cells. This process was also shown to enhance accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. The tests also showed that the process could be blocked by haloperidol and a DRD1 specific antagonist SKF83566, thus narrowing down the receptor to DRD1. Based on the experimental findings, the authors proposed an interaction model, shown in Figure 2. According to an explanatory accompanying paper, it has been hypothesised that TFH cells have a very stringent requirement for efficiency and specificity at the immunological synapse, which explains the finding that dopamine is used by human TFH cells, but not by human T cells of other subclasses.

So now I have some questions. Is there a possible mechanism where dopamine from a neuron stimulates B cells? Does this finding explain why in certain dopamine related disorders such as schizophrenia (where there is presumably an increased dopamine actvity) have an increased autoimmune phenomenon. As Hai Qi speculates, "When disease characteristics or treatment options are associated with changes in dopamine, the possible involvement of, and implications for, antibody- mediated immunity should be considered".

As Papa the lead author comments, “These particles were previously thought to only exist in neurons in the brain and we think they are, potentially, an excellent target for therapies to speed up or dampen the body’s immune response, depending on the disease you’re dealing with. Like neurons, specialised T cells transfer dopamine to B cells that provides additional ‘motivation’ for B cells to produce the best antibodies they can to help to clear up an infection. The human body has developed an advanced form of protection against bacteria, viruses and other foreign bodies that relies on the immune system".

References:

Papa I, Saliba D, Ponzoni M, Bustamante S, Canete P, Gonzalez-Figueroa P et al. TFH-derived dopamine accelerates productive synapses in germinal centres. Nature. 2017;547(7663):318-323.

Qi H. Immunology: Nervous crosstalk to make antibodies. Nature. 2017;547(7663):288-290.

Friday, July 21, 2017

nCD64 as a marker of Sepsis

Several times in my blogs, I have talked about how important it is to make a diagnosis at the fastest turn around time possible. In an attempt to miniaturise the testing platform and obtaining faster results, several technologies have been tested. In context with infections, genome detection and sequencing based technologies are increasingly becoming better and more accessible. Another example is pathogen specific molecular marker detection method on which a good lot of R&D is invested. MALDI-TOF is an excellent example.

Fig 1: Hospitalisation rates for sepsis or septicemia.
Sepsis is a serious issue. Any clinical microbiologist who works in association with the hospital knows the seriousness of sepsis. The terms "Sepsis" and "Septicemia" both refer to a bloodstream infection. Though in a strictly technical sense they mean two different things, they have been interchangeably used in literature and was widely accepted as similar. The earlier definition of sepsis was based on the idea that it is a systemic response and was thus assessed using a systemic inflammatory response syndrome (SIRS) criteria. To date, there is no clear definition of what sepsis is though it is generally agreed that it means circulating pathogen in blood. The diagnosis is based on evidence of fever, respiratory rate and abnormal total WBC count followed by bacterial identification from blood culture. There are no global estimates of sepsis prevalence. Available estimates suggest a range of <1% in a population. However,  there is a significant trend observed everywhere as shown in Fig 1.

In most parts of the globe, a prediction of sepsis is made based on markers such as C reactive protein and procalcitonin levels. Many studies have attempted to come up with a marker. Some of the well-researched markers of sepsis include triggering receptor expressed on myeloid cells-1 (TREM-1), azurocidin, CD64, CD11b etc.

Fig 2: Process schematic of the differential expression-based
cell-counting technology. Source
Studying these markers in the laboratory is not the big deal, since instruments such as Flow cytometers and other sophisticated equipments can do it. But they are not ideal for POCT (Point of care testing). In 2015, this problem was addressed by developing a POCT equipment based on microfluidics. The same group has now come up with improvements in design. The microfluidic biochip is capable of enumerating leukocytes and quantify neutrophil CD64 (nCD64) levels from 10 ml of whole blood without any manual processing. The tech uses whole blood (10ml) which is pumped into the biochip along with lysing and quenching buffers, to lyse erythrocytes. Cells are electrically counted and differentiated based on size using microfabricated electrodes. The CD64+ cells get captured based on their CD64 expression level. The difference in the cell counts is used to calculate nCD64 expression level. See Fig 2.

The authors claim that this technology can have profound results since the assay takes about 30 min and has scope for further improvement. That would be something really usefull to clinicians as a bedside tool for identifying sepsis.

References:

Mervyn Singer et al. The Third International ConsensusDefinitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016;315(8):801-810. doi:10.1001/jama.2016.0287

Mayr F, Yende S, Angus D. Epidemiology of severe sepsis. Virulence. 2013;5(1):4-11.

Wang X, Li ZY, Zeng L, Zhang AQ, Pan W, Gu W, Jiang JX. Neutrophil CD64 expression as a diagnostic marker for sepsis in adult patients: a meta-analysis. Crit Care. 2015 Jun 10;19:245. doi: 10.1186/s13054-015-0972-z.

Hassan U, Reddy B Jr, Damhorst G, Sonoiki O, Ghonge T, Yang C, Bashir R. A microfluidic biochip for complete blood cell counts at the point-of-care. Technology (Singap World Sci). 2015 Dec;3 (4):201-213. DOI: 10.1142/S2339547815500090

Hassan U et al. A point-of-care microfluidic biochip for quantification of CD64 expression from whole blood for sepsis stratification. Nat Commun. 2017 Jul 3;8:15949. doi: 10.1038/ncomms15949.

Monday, July 03, 2017

Lab series #17: Labelling methods for Quantitative Proteomics by MS

In an earlier post, I have talked about the principle of how a mass spectrometry works (Link) and how proteomics by sequencing is done using MS (Link). I had a few readers who suggested the idea that I have talked about MS-based shotgun sequencing but proteomics could be done even without sequencing. For example, MALDI-TOF analysis can tell about the protein identity which doesn't involve sequencing. This is absolutely true. However, such assays are now nearly outdated and sequence information can give us a lot more insight than just predicting protein based on the m/z values. In the earlier post, I ended with a note saying that I will revert back to the topic and talk about proteogenomics, targeted proteomics and quantitative proteomics. In this post, I will talk about labelling methods for quantitative proteomics or sometimes referred to as differential proteomics. If you have not read my earlier posts on MS, I strongly recommend that you read them first.

Let us build an example scenario. You want to learn what are the changes that occur in the cell after a virus infection. The most likely scenario in terms of proteome would be certain proteins will have increased expression and certain will have decreased expression, as a result of interaction with a virus. If you could find out what those proteins are, then there is a good chance that you could predict the pathways that have been disrupted. But for identifying what is the fold change, we have to quantify each protein. In a traditional assay like quantitative ELISA, the protein is directly estimated using a set of standards and then plot a graph. In proteomics, several thousand proteins are estimated in a single run and hence it is not practical to have several standards for every individual protein.  MS technique is originally designed to be a detection methodology and not a quantitative technique.

MS is a very sensitive technique, and there is a statistical chance that certain ions are more easily picked up than others which mean that the peak height or area in a mass spectrum in itself does not accurately reflect the abundance of a peptide in the sample. The main reasons for this are the differences in ionisation efficiency and detectability of peptides. Mathematically the equation would look something like this (I will not get into the actual mathematics since that is not relevant here).

Protein concentration= MS abundance value x Error factor

The error factor depends on each run and will vary from experiment to experiment. Consider this experiment. If you have a cell lysate you run it 10 times in LC-MS/MS analysis the final result will be varied from experiment to experiment. In fact, the number of proteins identified will also significantly change and you can expect a variation of at least 30% between any two runs as shown by multiple studies. If you run 2 independent batches of LC-MS/MS for comparison then the final result will consist only of error for purposes of direct comparison. The best idea would be to compare proteins from test and control in the same run so that the error will be constant. Since the error factor is the same in both cases (which is unknown), relative fold change can be accurately calculated by comparing the abundance value of m/z peak from the experiment. 


So what is required for comparison is to run all the protein preparation that has to be compared in a single mass spec run. Now you need a method to tell which peptide came from whom. That is why we label the peptide library obtained from each case. Let us say you want to run 5 biological test cases against 5 biological control case that would be a 10 plex labelling experiment with each condition being labelled with a different label. The label will tell MS where the peptide originally came from and how much of it is there in t.

Fig 1: Hypothetical example of m/z abundance
as an indicator of fold change.
Fig 1, is a hypothetical example of m/z abundance as an indicator of fold change. Consider you are comparing 3 cases against a control sample. The height of the peak represents the peptide abundance. In comparison to control, the case 1 is slightly elevated, case 2 is drastically down and case 3 is unchanged. This kind of comparison is available for all the peptides that have been detected in MS. The overall finding is then curated by the software and presented as a protein expression data with reference to the control.

Fig 2: Labelling methods for quantification of proteins in Mass Spectrometry.
There are wide varieties of labelling methods available and different literature have a different classification and there is an overlap in some cases. For simplicity, labelling methods can be broadly classified into 3 subtypes- Metabolic, Enzymatic and chemical labelling.See Fig 2 for a summarised classification.  It is not possible to talk about all the methods and intricate details of every method, which would make this post too long. I will stick to explaining a few methods that are more famous in biological practice which will give an idea of what exactly is happening. Chemical labelling is much similar to metabolic labelling except that the label is chemically attached to a particular peptide after extraction unlike doing it metabolically. Enzymatic labelling is almost a chemical labelling except that it is done using an enzymatic process.

Stable isotope labelling by amino acids in cell culture (SILAC)

Fig 3: Example light and heavy amino acids for SILAC.
SILAC labelling was first demonstrated from Matthias Mann lab; 2002. The method is a metabolic labelling method. The core idea is that cells are given essential amino acids that carry heavy stable isotopes continuously, which gets converted into proteins in the cell. This process run for sufficient time, a great majority of the cell proteins contain heavy labelled isotopes which can be picked in the mass spec. In a typical SILAC labelling experiment, lysine and arginine residues are isotope labelled. Since trypsin digestion is commonly used for obtaining peptides this results in labelling of every peptide in the mixture. The labels are available as N terminal and C terminal labelled lysine or arginine. See Fig 3. In addition, leucine, tyrosine and methionine amino acids with incorporated isotopes have also been used as labels. SILAC method has a high efficiency but comes with inherent limitations. Other than the facts that it is time consuming and expensive it requires that the method uses a culture system only those that can be cultured are available to work with this method.

18 0 labelling


The methodology considers the idea of class-2 proteases, such as trypsin, to catalyse the exchange of two 16 O atoms for two 18 O atoms at the C-terminal carboxyl group of proteolytic peptides. Hydrolysis of a protein in H218O by a protease results in the incorporation of one 18 O atom into the carboxyl terminus of each proteolytically generated peptide. Despite its simplicity, the method is not in regular use owing to the difficulty in attaining a high labelling accuracy.

Labelling using Isobaric tags

Fig 4: Structure of TMT tags. Source
This is probably one of the most common labelling methods to be used. Let us take the example of TMT (Tandem mass tags). Labels are basically isobaric compounds (They have same net mass) with a peptide binding site.

Each chemical tag contains a different number of heavy isotopes in the mass reporter region, which gives a unique reporter mass during tandem MS/MS for sample identification and relative quantitation, a mass normaliser which adjusts for the mass and a reactive group.

I have limited the discussion on labelling methods to the basic essence to give you an idea of how the system works. I recommend you read the references to have a detailed picture of the process.

References:

Tabb et al.  Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography-Tandem Mass Spectrometry. J Proteome Res. 2010 Feb 5; 9(2): 761. doi: 10.1021/pr9006365

Ong S, Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nature Protocols. 2007;1(6):2650-2660.

Rauniyar N, Yates J. Isobaric Labeling-Based Relative Quantification in Shotgun Proteomics. Journal of Proteome Research. 2014;13(12):5293-5309.