Tuesday, January 28, 2014

NextGen vaccination: Needle free devices, Interleukin adjuvants


   I have visited the topic of vaccination in this blog many different times. Vaccines are the safest and cost effective solution for many different infectious diseases. Approximations state, vaccination has saved a minimum of 35-40% of world population from developing fatal conditions. Problems still does exist in creating a safe and effective vaccine for infectious conditions (such as Tuberculosis, Staphylococcus aureus, HIV, Plasmodium, Influenza etc ), though efforts are made to improve. Vaccination for HPV (Gardasil) is a classic example, of a successful vaccine campaigning against cervical cancer.

Fig 1: Inovio Vaccine delivery.
      I have detailed about various approaches in HIV vaccination Research, in my previous posts. One of them was about use of DNA plasmids targeting the gag, pol, and env proteins of HIV-1, delivered by electroporation through a special device electroporation device (Link). Their product is referred as Pennvax. The vaccine is a mixture of 3 expression plasmids encoding HIV-1 Clade B Env, Gag, and Pol. According to a study 89% of the subjects who received IL-12 DNA together with the PENNVAX(R) DNA vaccine delivered with electroporation produced a vaccine specific CD4+ or CD8+ T cell response compared to 67% who received the DNA vaccine alone without the IL-12 DNA. That' s a huge leap. The confidence in this breakthrough approach inspired the design of IL- 28 combination with multi-antigen hepatitis C DNA vaccine, INO-8000 trial. I have heard that some similar approaches have been planned for cancer immuno- therapeutics as well.

       With the same concept, IL 33 has been used to boost the HPV vaccine powers. As Dr. Kim puts it, "We are developing multiple DNA plasmid based cytokine and chemokine genes as immune activators and I am proud to say that Inovio has more of these immune activators in its pipeline than anyone else in the world. We have initiated human studies using other DNA-based cytokines and look forward to moving IL-33 into clinical trials in combination with our DNA vaccines". Source

Photo 2: Vaccine-Delivery Patch.
   From a age of delivering vaccines through syringes we are now converting into a time scale where we have vaccines mounted on the surface of a chip. The chip would hold several vaccines which can be directly delivered into intradermal region, shown to produce a better immune response. This is the first improvement in our vaccination technology. Electroporation device also works by precise delivery system as shown in Fig 1. The precise delivery increases antigen presentation and thus better and effective response.

         The second important factor is the use of adjuvants. There is still a debate as of how the adjuvants work. In simplest terms, adjuvants are substances that increases the immunological response to an antigen. You can find an in depth discussion of the adjuvant mechanism of action here. I want to note, the use of interleukins themselves as a booster of immunity is a very new concept.

      Taking example from above, Il 12 has been shown to induce improve specific response against HIV. This maybe explained by, IL 12 is a central regulator of the cell mediated immunity. Similarly, the use of IL 28 maybe explanied by, its role in immune defense against viruses, important factors such as ISGF3G (Interferon Stimulated Gene Factor 3), Mx proteins etc. The use of IL 33, which is involved in regulation of helper T cells and promotes type 2 cytokine functions can enhance effects of antiviral and anti neoplastic cell activity which may explain the success.

     Take home message is immunity is a very delicate issue. A lack of immune response in one type of vaccine is not a proof of non antigenicity. Probably we have to test the right cocktail, and interleukins seem to be good candidates.

Kalams SA etal (2013). Safety and comparative immunogenicity of an HIV-1 DNA vaccine in combination with plasmid interleukin 12 and impact of intramuscular electroporation for delivery. The Journal of infectious diseases, 208 (5), 818-29 PMID: 23840043

Villarreal D, Wise MC, Walters JN, Reuschel E, Choi MJ, Obeng-Adjei N, Yan J, Morrow MP, & Weiner DB (2014). Alarmin IL-33 acts as an immunoadjuvant to enhance antigen-specific tumor immunity. Cancer research PMID: 24448242

Kichaev G, Mendoza JM, Amante D, Smith TR, McCoy JR, Sardesai NY, & Broderick KE (2013). Electroporation mediated DNA vaccination directly to a mucosal surface results in improved immune responses. Human vaccines & immunotherapeutics, 9 (10), 2041-8 PMID: 23954979

Gherardi MM, Ramírez JC, & Esteban M (2001). Towards a new generation of vaccines: the cytokine IL-12 as an adjuvant to enhance cellular immune responses to pathogens during prime-booster vaccination regimens. Histology and histopathology, 16 (2), 655-67 PMID: 11332721

Saturday, January 25, 2014

Clostridium difficile on a note


Photo 1: Endoscopic visualization of
Pseudomembranous colitis.
    Almost 2 years ago, I had posted one of the first posts on Clostridium difficile and Fecal Transplantation (Link). In the past 2 years there has been a sudden burst of literature on C diff and antibiotic-associated nosocomial diarrhea (AAD). As I would sluggishly point out in my discussions, its a brand ambasador of Nosocomial problems along with other fellow agents like Peusdomonas and Acinetobacter. In this post, I want to comment on the C diff, challenging some common assumptions based on evidence. 

     The credit of first time identification, is awarded to Hall and O'toole, in 1935. It was almost ubiquitously accepted that they are colonizers especially in infants. Studies argued that it was capable of producing toxin, but was not much biologically relevant. It was the work of Tedesco etal, that showed, people treated with clindamycin nearly 21% developed diarrhea. Based on studies it was found to cause Pseudo membranous colitis, and came to be famous in literature as "clindamycin colitis".

Table 1: Tests for diagnosis of C diff infection.
     C difficile is obligate anaerobic, spore-producing, gram-positive bacillus belonging to Clostridium taxonomy. Generally pathogenesis is considered under 3 steps- Alteration of normal flora by antibiotics, acquisition of a toxigenic strain, progression to clinical condition. The clinical condition is diagnosed by direct culture and detection of organism or by establishing the presence of toxins. I have outlined the choice of tests available in Table 1. The most commonly used test is ELISA for detection of toxin A and B.

       Often in literature, it has been pointed that the C diff toxigenic strain is acquired in hospital (Hence Nosocomial origin). A study published in NEJM, showed a genetically diverse sources for infection, suggesting that hospital by itself is not the only part of the source. Probably, people are constantly invaded by the strains. However, having a good normal flora defeats the C diff from making an impact.

      This understanding meets an important question. Often I have asked the question, can antibiotic by itself stimulate toxin production through some genetic response element. It has been noted that C diff toxin production is enhanced by antibiotics that disrupt anaerobic normal flora (such as clindamycin, ceftriaxone), and the one's which didn't attack anaerobic flora didn't have much impact. A larger proportion of gut flora is anaerobic, and hence my presumption is that the loss of competing anaerobic flora allows establishment. It has also been shown that antibiotics such as ampicillin and clindamycin can increase colonization by enhancing the expression of genes encoding colonization factors. bThe exact mechanism of how C diff influences is much a subject of study. A paper published in nature, studied how antibiotic influence the gut metabolome and thus influences infection. This is an example of priming for the infection. Unlike other scenario where the organism conditions the system for propagation, here the condition dictates the organism.

     There is a great deal of anecdotal evidence that people especially from southern part of India are less susceptible to C diff infections. This maybe due to consumption of milk and milk products, especially curds that are rich in Lactobacillis species. The same effect has also been shown in laboratory studies, where the presence of lactobacillus reduced the severity of C difficile.

    So what are the options? Clinically treatment with vancomycin was considered a standard, but recurrence was common. Fidaxomicin was approved for C diff Infections by FDA in 2011 and remains the standard. Other options include, Fecal transplant and probiotics. In the west, C diff has been more serious problem and a vaccine search is on. It is known that people with IgG antibodies directed against TcdA are protected and remain asymptomatic to infection. Sanofi Pasteur is currently onto a Phase III trial , using a vaccine toxoid (Link). Another novel vaccine attempt has been made by use of harmless spores (link) and is expected to work well (Link).

Bartlett JG (2008). Historical perspectives on studies of Clostridium difficile and C. difficile infection. Clinical infectious diseases, 46 Suppl 1 PMID: 18177220

Eyre DW etal (2013). Diverse sources of C. difficile infection identified on whole-genome sequencing. The New England journal of medicine, 369 (13), 1195-205 PMID: 24066741

Pultz NJ, & Donskey CJ (2005). Effect of antibiotic treatment on growth of and toxin production by Clostridium difficile in the cecal contents of mice. Antimicrobial agents and chemotherapy, 49 (8), 3529-32 PMID: 16048976

Denève C, Deloménie C, Barc MC, Collignon A, & Janoir C (2008). Antibiotics involved in Clostridium difficile-associated disease increase colonization factor gene expression. Journal of medical microbiology, 57 (Pt 6), 732-8 PMID: 18480330

Wednesday, January 15, 2014

PDIM: Yet another virulence factor of TB


       If you have noted from my posts, I have been often talking about a TB especially in the last few posts. Recently as a part of my work, I have been talking to a person working on TB. One of his comment was that there is a lot of literature available on Tuberculosis. But a lot less is understood. Other than HIV and Influenza, TB would be yet another candidate that will keep the scientists the busy for years to come. Previously, I have noted that improvements have been made in form of new class of drugs (Link), search for vaccines etc (Link). However, the understanding of the infection process is still a big halo.

Fig 1: PknG blocks phagosome-lysosome fusion.
         If I had ask you to explain the pathogenesis of tuberculosis in its simplest format how would do that. It would be something like this. Inhalation of infective droplet, bacterium reaches alveolus, engulfed by alveolar macrophages, resist intracellular killing, multiplication, spread and infection. That seems so simple. But notice, unlike many other pathogens that you studied in medicine (a character shared by only a few), this bacteria has the ability to stay inside the "defender cells" (such as macrophages). Tubercle bacilli may stay dormant in CD271+ mesenchymal cells. The bacilli can actually survive inside the phagosome by blocking the formation of an active phagolysosome. This is achieved by the bacterium PknG which block phagosome-lysosome fusion. The bacteria can also block vacuolar-H–ATPase inhibiting phagosome acidification using PtpA (Protein Tyrosine Phosphatase).

      It has been very much clear from experiments and anecdotal findings that this isn't part of the full story. How does the bacteria coordinate this whole survival strategy. Notice from my above paragraph, the bacteria by itself doesn't invade the pulmonary tissue. It is the ability to survive inside the macrophages that carries them into deeper tissue, by a Piggy-back mechanism (You get my point).

     First question that is asked would be, is Piggy-backing the tubercle bacilli just a matter of cell carrying it anywhere the cell wants to, or somehow directed by the bacilli itself. The latest study shows that perhaps it is pathogen directed. The study established that the pathogenic TB can recruit permissive macrophages to the site using a chemokine receptor 2 (CCR2)-mediated pathway. Furthermore it evades immunity with the help of a cell-surface-associated PDIM (Phthiocerol Dimycoceroserate) lipid which shields the underlying PAMPs. The paper challenged the common idea that alveolar surfaces of the distal lung offer a more favorable environment for mycobacterial proliferation. Instead what the study shows is that other members of normal flora can get the TLR signalling right which leads to clearing of organism. In contrast, alveolar micro-environment is sterile and thus gets the bacteria ample chance to multiply.

    That leads me to think a couple of thoughts. Is there a similar comparable mechanism in Salmonella causing enteric fever, cause they too take a ride by resisting intracellular killing. 2nd question, If i could unmask the PDIM coat by perhaps inhibiting its synthesis, would that be the wonder drug for TB? Perhaps more research can tell

Das B, Kashino SS, Pulu I, Kalita D, Swami V, Yeger H, Felsher DW, & Campos-Neto A (2013). CD271(+) bone marrow mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis. Science translational medicine, 5 (170) PMID: 23363977

Warner DF, & Mizrahi V (2007). The survival kit of Mycobacterium tuberculosis. Nature medicine, 13 (3), 282-4 PMID: 17342138

Wong D, Bach H, Sun J, Hmama Z, & Av-Gay Y (2011). Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+-ATPase to inhibit phagosome acidification. PNAS, 108 (48), 19371-6 PMID: 22087003

Cambier CJ, Takaki KK, Larson RP, Hernandez RE, Tobin DM, Urdahl KB, Cosma CL, & Ramakrishnan L (2014). Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature, 505 (7482), 218-22 PMID: 24336213

Kugelberg E (2013). Immune evasion: Mycobacteria hide from TLRs. Nature reviews. Immunology. doi:10.1038/nri3604

Monday, January 06, 2014

Bacterial Persistence- On a Note


     Before I say anything, I wish all a happy New Year (Oh, Yes, am a couple of days late wishing). Am slowly entering the 3rd Year of blogging with hopefully considerable improvement in the material. Probably the person who has gained the most from this blog is myself. It reals helps me to write down my thoughts somewhere. The downside, I have been pretty inconsistent in posting last year. Hope I can make up this year.

        Often i get into arguments with people on what mechanisms influence antibiotic resistance. Acquired antibiotic resistance is one of the important problems in chemotherapy (But not the Only). The kinetics of acquiring resistance is often quite complex. The long standing idea is there is a pre-existing mutation in one or few of the organisms that is being selected for when antibiotic is given leading to emergence of a resistant strain. That is a well credited idea. But, a couple of mutations most often will not lead to a huge leap in MIC for organism in one single replication event. As I have argued before, and still continue to debate, it requires something extra.

    Here's one scenario that often strikes me to project an explanation. Clinical Microbiologists practicing in routine patient care may have noted the following. A disc diffusion test (Usually Kirby Bauer Method), shows sensitivity to a X antibiotic. When the clinician uses the same antibiotic the patient doesn't improve. If you collect sample and isolate again, you still get sensitivity in plate to X. This is not a very common phenomenon, but not so rare either. I simply mean to state, the organism shows sensitivity in vitro and resistance invivo. Both case may be right at the same time, and technically correct.

Example mechanism of persistance. Read more here
   My explanation was that there maybe a "Bystander resistance". But there seems to be a more legitimate alternative explanation and probably, a very unexplored area. In many of my previous blog posts, I had mentioned of existence of subset of population called as persisters. Persisters are a small subgroup of the population that undergoes dormancy, making them highly resistant to the activity of drugs. The concept of persisters has been proposed almost a decade ago, but is probably taken seriously only recently. An excellent commentary is presented by Andrew Jermy.

     On a lighter note, I wish to say that gone are the days when we used to think that and teach there are 2 mechanisms of antibiotic resistance- Innate and Acquired. There are more mechanisms which doesn't fall into any of the classic categories.

Wakamoto Y, Dhar N, Chait R, Schneider K, Signorino-Gelo F, Leibler S, & McKinney JD (2013). Dynamic persistence of antibiotic-stressed mycobacteria. Science (New York, N.Y.), 339 (6115), 91-5 PMID: 23288538

Jermy A (2013). Bacterial physiology: no rest for the persisters. Nature reviews. Microbiology, 11 (3) PMID: 23334264

Wood TK, Knabel SJ, & Kwan BW (2013). Bacterial persister cell formation and dormancy. Applied and environmental microbiology, 79 (23), 7116-21 PMID: 24038684