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.