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.

Sunday, July 02, 2017

Angiostrongylus cantonensis

By the end of April this year, rat lungworm or Angiostrongylus cantonensis raised an alarm with 6 cases which was apparently spreading on the Hawaiian island of Maui. The infection is called as angiostrongyliasis. it basically manifests as meningitis and if not treated can cause permanent damage to the central nervous system or death. A cantonensis is well known in South-east Asian and Pacific region and is one of the most common causes of eosinophilic meningitis. The More recently, the parasite is been found in environments of California, Alabama, Louisiana Leon and Florida regions including Alachua, Saint Johns, Orange, and Hillsborough (Where it is much more common than originally realised).

Photo 1: Adult female of A cantonensis.
Source
A cantonensis is a species related to Strongyloides stercoralis (Order strongylida, and superfamily metastrongyloidea) was first described from CSF of an eosinophilic meningitis case by Nomura and Lim in Taiwan in 1944. Morphologically, they appear like other nematodes unsegmented with a simple fully developed gastrointestinal system. Males are smaller than the females. Photo 1, shows an adult female with a dark red digestive organ, two white reproductive organs, and a transparent cuticle.

Photo 2:cantonensis egg.
Source
A cantonensis is classically an infection of rats and humans are accidental hosts. A cantonensis reside in pulmonary arteries of rats. A female lays up to 15000 eggs a day. Once eggs are laid, they hatch and the first stage larva migrates up the pharynx and are swallowed to be secreted through faeces. Snail forms an intermediary host which is infected with the larva where it develops. The 3rd stage larva in the intermediate host is the infective stage. It enters through contaminated water, ingestion of snails the larva migrates up the brain. The larvae develop into a pre adult stage and then return to the pulmonary artery, where it matures.

Fig 1: Life cycle of A cantonensis.Source
Many species of snails and slugs serve as intermediate hosts for A cantonensis. Some of the well known vectors include Achatina fulica (African land snail), Pila species, golden apple snail etc

Humans acquire the infection accidentally through ingestion of contaminated water, other infected paratenic animals (crabs, freshwater shrimps) or leafy vegetables which are not cleaned and washed well.Fig 1 from CDC page gives a detailed picture of the life cycle.

The clinical presentation will usually involve symptoms such as abdominal discomfort which progress to fever and headache. Often the case will self resolve without treatment and may not be evident. In cases of high load of parasites, the infection progresses to serious meningitis. The laboratory diagnosis is at best, a prediction. Eosinophilic meningitis and imaging studies maybe a clue. To date, there are no definitive diagnostics available. It should be noted that eosinophilic meningitis is not a definitive indicator for A cantonensis. Other agents include Angiostrongylus costaricensis, Gnathostoma spinigerum etc. Though PCR testing is available it is done only in special labs and not widely available.

In a recently published article in Digital Journal by Karen Graham, there is a discussion on what increased identification of A cantonensis means in Florida. As Heather Stockdale Warden comments, "The ability for this historically subtropical nematode to thrive in a more temperate climate is alarming. The reality is that it is probably in more countries than we found it in, and it is also probably more prevalent in the southeastern U.S. than we think".

References:

Stockdale Walden, H., Slapcinsky, J., Roff, S., Mendieta Calle, J., Diaz Goodwin, Z., Stern, J., Corlett, R., Conway, J. and McIntosh, A. (2017). Geographic distribution of Angiostrongylus cantonensis in wild rats (Rattus rattus) and terrestrial snails in Florida, USA. PLOS ONE, 12(5), p.e0177910.


Wednesday, June 07, 2017

Superdrug for the Superbug

Antibiotic resistance is a huge problem and the problem of so-called "Superbugs" is not new to the readers. There are several reports of resistance against drugs that are considered as reserved only as last resort. Colistin is an example, which is preferentially used against gram negatives as the last drug of choice. Similarly, Vancomycin is a reserved antibiotic for gram positives, especially MRSA. MRSA now being an absolutely common isolate vancomycin is more commonly used.

Fig 1: Mechanism of Vancomycin resistance. Source
Vancomycin (Initially code-named as 05865 and later marketed as Vancocin) was first isolated by Edmund Kornfeld in 1953 from a soil sample collected from Amycolatopsis orientalis from the interior jungles of Borneo. The toxicity was a limiting factor and antibiotic was reserved only for those cases, where nothing else worked. Chemically, Vancomycin is a glycopeptide (branched tricyclic glycosylated nonribosomal peptide) and acts by inhibiting cell wall synthesis. Vancomycin binds to the terminal D-Ala-D-Ala dipeptide which halts the process of peptidoglycan formation. The resistance is due to change in D-Ala-D-Ala. The most common change is D-Ala-D-Lac which has orders of low binding of vancomycin. See Fig 1, for details.

Vancomycin resistance has been a huge problem in health care settings. There are several genes that mediate resistance to vancomycin named as VanA, VanB, VanC, VanD and VanE. The commonly talked about vancomycin resistant strains are VRSA (Vancomycin resistant Staphylococcus aureus) and VRE (vancomycin resistant Enterococcus species). These are very difficult to treat infections. The most common mechanism of glycopeptide resistance is to develop modified cell membrane receptors with reduced affinity. The problem has been recognised quite early and one of the obvious methods of dealing with it is to modify the vancomycin. As early as 1999, a collaborative group between Princeton and Merck showed that the carbohydrate derivatives of vancomycin were able to overcome resistance to vancomycin.

Fig 2: Structure of[Ψ[C(═NH)NH]Tpg4]vancomycin
aglycon. Source
There are several reports in the literature reporting successful modifications of vancomycin increasing its kill capabilities. Many of these were loosely called as Vancomycin ver 2.0. In 2011, Dale Bogger's lab from Scripps Institute reported the development of a new vancomycin derivative a [Ψ[C(═NH)NH]Tpg4] vancomycin aglycon which could bind both d-Ala-d-Ala and d-Ala-d-Lac. 

Fig 3: Structure of modified vancomyin 3.0. Source
Improving on their work Bogger's lab has now reported the design of a Vancomycin 3.0 (CBP  C1- aminomethylene vancomycin). This chemical has 3 different modes of activity. Not only it has a dual binding activity, the addition of quaternary ammonium salt in the structure (It was a calculated design), provided a binding pocket-modified vancomycin analogue which was independent of D-Ala-D-Ala or D-Ala-D-Lac binding. Another modification of peripheral (4-chlorobiphenyl) methyl (CBP) to the vancomycin disaccharide made it highly potent. The same drug also can induce cell permeability. Basically, the same drug has 3 independent mechanisms of action, making it difficult for the bacteria to acquire resistance. Indeed, there is data to show that in vitro the bacteria didn't acquire any resistance even after 50 passages.

As Bogger States, "Organisms just can't simultaneously work to find a way around three independent mechanisms of action. Even if they found a solution to one of those, the organisms would still be killed by the other two".

The compound is not yet ready for human clinical use. The chemical synthesis is very laborious and it is not known as to what are its side effects. 

References:

Levine D. Vancomycin: A History. Clinical Infectious Diseases. 2006;42(Supplement 1): S5-S12. 

Ge M. Vancomycin Derivatives That Inhibit Peptidoglycan Biosynthesis Without Binding D-Ala-D-Ala. Science. 1999;284(5413):507-511.

Xie J, Pierce J, James R, Okano A, Boger D. A Redesigned Vancomycin Engineered for Dual d-Ala-d-Ala and d-Ala-d-Lac Binding Exhibits Potent Antimicrobial Activity Against Vancomycin-Resistant Bacteria. Journal of the American Chemical Society. 2011;133(35):13946-13949.

Okano A, Isley N, Boger D. Peripheral modifications of [Ψ[CH 2 NH]Tpg 4 ]vancomycin with added synergistic mechanisms of action provide durable and potent antibiotics. PNAS. 2017;:201704125.

Tuesday, May 30, 2017

Ebola 2017

Photo 1: Ebola Poster. Source
Ebola epidemic has been the point of discussion in many of my past blog posts. Considering that the previous lethargic early response was met with a devastating outbreak, the global health community is currently monitoring a possible resurgence of the Ebola virus in the Democratic Republic of Congo (DRC). As of on May 26, 2017, I gather from reports that there have been at least 43 cases of suspected Ebola. Of these only two cases have been positively confirmed in a laboratory. There are 4 probable and 16 suspected cases and a number of approximately 400 contacts. The reported cases currently remain confined to Likasi. Regions of hot interest are currently Nambwa, Muma, Ngayi, Azande, Ngabatala, Mobenge and Mabongo. This is the 7th documented outbreak of Ebola in the DRC since the discovery of the virus in 1976.

Fig 1: Likasi, Democratic Republic of Congo.
Source 
The first case (Most probably the index case), was a 45-year-old hunter male seen on 22 April 2017 from Bas Uele Province in the north of the Democratic Republic of the Congo (DRC), bordering the Central African Republic. Currently, nothing much is known about the genetics of the strain and the outbreak appears to be very limited by geography. Based on an RT-PCR from one of the isolate, it appears that the virus is related to Zaire subtype.

A very interesting perspective is that the Ebola this time has the possibility of involvement of Pig. Though such a link is not yet established, here is the argument. A smoking gun is that 84 pigs have recently died in the epicentre of the current outbreak. There is also some possible evidence that Ebola- Zaire strain can infect and manifest in pigs and can be transmitted between them. They can further transmit it to primates. A counter thought is that considering huge deaths of pigs, experts suggest that there should have been more human cases. As epidemiologist Fabian Leendertz comments, “I’m doubtful that the pigs actually carry Ebola, but we have to test them".

Photo 2: Merck Ebola Vaccine
Recombinant vesicular stomatitis virus–Zaire Ebola virus (rVSV-ZEBOV) is a product of Merck, which was successfully tested earlier in 2015- 2016. The vaccine consists of vesicular stomatitis virus (VSV), which has been genetically engineered to express a glycoprotein from the Zaire Ebolavirus leading to a neutralising antibody immune response

The earlier clinical trial showed that of the 5837 people who received the vaccine, no Ebola cases were recorded 10 days or more after vaccination. In comparison, there were 23 cases 10 days or more after vaccination among those who did not receive the vaccine. That's a 100% coverage. However, questions were raised since the trial was conducted during the dying phase of the epidemic. It is also not know how long does the immune memory stays. The vaccine is unlicensed and ethics committee at the DRC has approved a clinical study of rVSV-ZEBOV Ebola vaccine, in the hope that it would arrest the outbreak.

References:

Ebola Virus Disease - Democratic Republic of the Congo: External Situation Report 14 (28 May 2017). Published on 28 May 2017. Link

Kai Kupferschmidt, Jon Cohen. Could pigs be involved in Congo's new Ebola outbreak? Link

Henao-Restrepo A, Camacho A, Longini I, Watson C, Edmunds W, Egger M et al. Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ça Suffit!). The Lancet. 2017;389(10068):505-518.

Friday, May 26, 2017

Understanding Anelloviruses

Recall that I have recently written a post on human blood virome and one of the major findings was that there are a lot of viruses in human blood circulation that is apparently normal. A major critique of the study is that there are a lot of contamination issues to be ruled out. It is really not clear as to if the virus is actually in the blood, or has been introduced from the skin during the collection procedure. If you look at the past literature on WGS technology it is undisputedly clear that in most cases skin microbiome is introduced during blood collection even when sterile precautions are taken, at a non-negligible level. One virus that was particularly striking was Anellovirus, something not many people have heard about. That is the point of discussion here.

Photo: Torque teno virus.
Anellovirses are non-enveloped, icosahedral symmetry virus (T=1) with a genome containing a circular single-stranded DNA coding for 3 major open reading frames (ORFs). In addition, they carry several small ORFs and untranslated regions (See Fig 1). Torque teno virus (TTV), is the most studied member of the group which was first reported in 1997 in a Japanese patient post-transfusion. 2 more virus that is now commonly talked about includes Torque teno mini virus (TTMV) and Torque teno midi virus (TTMDV). Subsequently, there have been several different studies on Anellovirus in blood and blood derived products. In all of the studies, Anelloviruses have been found in varying percentages.

Fig 1: Genome organization of
Torque teno virus. Source
It is my understanding that Anelloviruses was previously described as under the family Circoviridae, genus Anellovirus. Currently, Anellovirus itself is the family and have several genera under them. See Table 1 for a compiled information on the classification details.

Anellovirus infection though earlier thought to be more of blood-related have been identified as ubiquitous and estimates are that as much as 90% of global population harbours Anellovirus. This can be seen by detecting the sequences in at least one of the biological samples such as blood, skin, saliva, gut etc. Further, these are not human specific and Anelloviruses have been found in other species such as human primates and wide variety of domestic animals. 

Table 1: Anellovirus classification.
Anelloviruses have been implicated as a correlation with conditions such as fever and associated with hepatitis virus infections. However, they appear to be more of a random phenomenon and no study has been published implicating them as a significant contributor and modulator of a clinical condition. For the time being at least, they appear to be floaters (Commensals) and nothing much of their biology is known.

References

1. McElvania TeKippe E, Wylie K, Deych E, Sodergren E, Weinstock G, Storch G. Increased Prevalence of Anellovirus in Pediatric Patients with Fever. PLoS ONE. 2012;7(11):e50937. 

2. Al-Qahtani A, Alabsi E, AbuOdeh R, Thalib L, Nasrallah G. Prevalence of anelloviruses (TTV, TTMDV, and TTMV) in healthy blood donors and in patients infected with HBV or HCV in Qatar. Virology Journal. 2016;13(1).

3. Spandole S, Cimponeriu D, Berca LM, Mihăescu G. Human anelloviruses: an update of molecular, epidemiological and clinical aspects. Arch Virol. 2015 Apr;160(4):893-908

Wednesday, May 17, 2017

VPM1002 TB vaccine to be tested in India

Tuberculosis continues to be a high burden problem in many parts of the world especially, its co-infection with HIV creates substantial complications. Though BCG is universally administered, BCG fails to protect after a certain number of years. Research is currently focussed on inventing a totally new vaccine or to create modifications in BCG allowing better vaccine performance. In India, a recombinant vaccine called VPM1002 is planned to be tested for phase II/III vaccine trial. Here are some details of this vaccine.

Fig 1: Schematic description of the underlying
mechanism of improved T cell stimulation
by VPM1002. Source
BCG is derived from M bovis and has immunological properties which is much similar to M tuberculosis. The bacteria is phagocytosed by host macrophages, and are efficiently trapped in cellular phagosome. However, the cell fails to digest the bacteria due to multiple virulence mechanism of the bacteria. One of them is a urease which plays a role in pH neutralization of phagosome thus denying its maturationSubsequently, antigens are processed by MHC II pathway and induce CD4+ T-cell responses but what is actually required is a CD8+T cell response. In 2005 a JCI paper reported a rBCG (Recombinant BCG) prague strain that secretes listeriolysin of Listeria monocytogenes and is urease C-deficient. The idea being that the a urease deficient mycobacterium secreting listeriolysin allows better phagosome maturation and potentially activating MHC I pathway thus eliciting a desired CD8 response. Vaccine developed from this strain is called as VPM1002. Prof. Kaufmann comments “The vaccine being tested is intended to replace the current BCG vaccine and will be administered to young children to protect them against tuberculosis. Adults may also be able to benefit from it later”. In a phase I Clinical trial conducted in Germany  and and South Africa, volunteers were followed up for 6 months after a single vaccination with 5 x 10 CFU was safe, well tolerated and induced multifunctional CD4+ and CD8+ T-cells. Studies have also showed that rBCG is significantly better in terms of immunity and safety. The findings were subsequently confirmed by a phase 2a study in South Africa.

Photo: VPM1002 Vaccine.
Source
India being a high burden for TB desperately needs a new and better vaccine. Serum Institute of India has apparently obtained approval to go ahead with Phase II/III vaccine testing. This will be a double blind placebo-controlled, randomised clinical trial which will see enrolment of 2,000 adults (1000 participants will receive vaccine and the other 1000 will receive placebo) in 17 centres. A single dose will be administered and followed up for 12 months. The work is planned in 2 phases. In the 1st phase, 200 participants will be given the vaccine and safety evaluated. In the second phase (given the success of first phase), the remaining volunteers will be vaccinated. Dr. Prasad S. Kulkarni, Medical Director at Serum Institute says, “Adults who have completed TB treatment will be first screened and enrolled if found eligible 2-4 weeks after completion of TB treatment. Traces of the drugs may be present in the body for two weeks after completion of the treatment. Since the vaccine contains live, weakened bacteria, the drugs can kill them if given earlier than two weeks after completing the treatment.”

The key importance of VPM1002 in contrast with BCG is superior immunity conferred by CD8+ T cells, enhanced Il-17 secretion. It also reduces the risk associated with BCG complications seen in a small subgroup of HIV positive subjects.

References:

Grode L. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin. Journal of Clinical Investigation. 2005;115(9):2472-2479.

Grode L, Ganoza C, Brohm C, Weiner J, Eisele B, Kaufmann S. Safety and immunogenicity of the recombinant BCG vaccine VPM1002 in a phase 1 open-label randomized clinical trial. Vaccine. 2013;31(9):1340-1348.

VPM Study group. HIV-unexposed newborn infants in South Africa in HIV-unexposed newborn infants in South Africa. Clin. Vaccine Immunol. doi:10.1128/CVI.00439-16

Tuesday, May 09, 2017

Atlas Map for blood Dendritic cells, Monocytes, and Progenitors

I note that I have been unable to post frequently in past few weeks, since I have been very busy with work and personal travel. I have been getting a few mails enquiring if I moved to a new page. No, I havent yet. If I do, I will definetly make an announcement.

Immunology is still a very difficult subject especially when it comes to classification. The problem arises from the lack of understanding and partly cause classification system is messy. Further, the same immune cell function differently under different circumstances. Cellular subtyping and subsequent studies are based largely on the established classification rules, which has been developed decades ago. In most cases, a subtypes is based on morphology and a little understanding of function. For most of the human cells, there is no clear cut systematic cellular classification based on molecular profile. Human cell atlas in one of the ambitious project that has been announced to overcome this massive question. Sarah Teichmann states, “The cell is the key to understanding the biology of health and disease, but we are currently limited in our understanding of how cells differ across each organ, or even how many cell types there are in the body. The Human Cell Atlas initiative is the beginning of a new era of cellular understanding as we will discover new cell types, find how cells change across time, during development and disease, and gain a better understanding of biology.”

The methodology used for this is technically complex but the idea is straightforward. The cells are sorted out using a known classification system and then each cell is isolated from the pool. Then using a technique called single cell transcriptomics a cell is profiled. This gives a picture of what is the genetic expression pattern of that particular cell. By parallely doing so for a huge number of cell samples, a map can be drawn.

Dendritic cells are called so because of their structure. They are one of the most important classic APC (Antigen presenting cell). Dendritic cells (DC) were first discovered by Ralph Steinman roughly 4 decades ago for which he was awarded the Nobel prize. Though there has been some previous hints that DCs are a diverse population, the subtypes have not been recognised or typable. 


In the study by Villani et al human PBMCs were enriched from blood sample using Ficoll extraction and DCs were isolated using flow cytometry. Each cell was then isolated into a single well and transcriptome analysis was done. For the study, 742 DCs and 339 monocytes that passed quality control was profiled with an average detection of 5326 genes per cell. Based on genetic expression cluster results 6 clusters which are numbered DC1 to DC6 were identified (See Fig 1 for classification details).
  • 2 clusters mapping closely to the well-established DC subsets, with cluster DC1 mapping to CD141+ DCs and cluster DC6 to pDCs.
  • 2 clusters containing the CD1C+ cDCs, cluster DC2 and cluster DC3
  • 1 cluster corresponding to CD141–CD1C– population named DC4
  • 1 cluster DC5
Fig 1: Summary of the study and revised classification of the Dendritic cells to include 6 different subtypes based on the transcriptomic map. Source
Further analysis was able to gathered some potential markers of interest.
  • DC1 corresponds to CD141/BDCA-3+ ,which was best indentified using CLEC9A.
  • DC2 and DC3 togther corresponds with CD1C/BDCA-1+
  • DC4 corresponded with CD1C–CD141–CD11C+ which also shared some signatures with monocytes
  • DC5 was unique in itself.
  • DC6 corresponded to the interferon-producing pDC, defined by standard markers CD123, CD303/BDCA-2
The paper is very dense with lot of data. However, essentially this paper established that there are DC subtypes defined and probably there is a diversity in function. This also sits well with literature where function of DCs is called into into three categories. First, antigen presentation and activation of T cells. Second, inducing and maintaining immune tolerance. Third, maintaintainence of immune memory. However, which does what is yet to be studied and elucidated. Divya Shah comments, “In this study, scientists have used cutting-edge technologies to find that there are many more types of cell than we originally thought. The next step is to find out what each of these cell types do in our immune system, both when we’re healthy and during disease."

Reference

Villani A, Satija R, Reynolds G, Sarkizova S, Shekhar K, Fletcher J et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science. 2017; 356 (6335): eaah4573.

Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors