A new mechanism of immune evasion by Leishmania parasites

Posted by Kasra

It is always pleasant to read papers published by friends and old colleagues.  Also it was about time I would write another post about Leishmania. This new paper published in Cell host and microbe (pubmed index) discusses a new mechanism that Leishmania parasites use to evade the host immunity.

 A quick reminder, the promastigote forms of Leishmania enter the mammalian host via bite of the sandfly. They are readily phagocytosed by macrophages. Yet, they manage to evade to propagate within the macrophage thanks to multiple mechanisms of immune modulation and evasion. 

Previous publications of this group that showed how Leishmania is able to impair maturation of the phagolysosome (for example delay acidification) by interfering with phagolysosome associated proteins. Their previous work had pointed to Leishmania‘s surface lipophosphoglycan LPG. Following their work on the phagosome, they look to see if there are other molecules that are also altered after Leishmania infection, stumbling upon VAMP3 and VAMP8, two SNAREs that are cleaved after infection. They find that this time this time this cleavage is due to another important virulence factor of Leishmania namely surface protease GP63.  This protease has been shown to have many immunomodulatory properties and cleaving many important macrophage proteins (phosphatases, transcription factors…) and now there is a new one on the list.  The authors show that VAMP8 is important for cross-presentation of antigens from MHCII to MHCI. To show this they create ovalbumin expressing expressing L. major and see how presence or absence of GP63 could affect activation of OT-II (ovalbumin-reactive T cells) after coculture with macrophages. Importance of VAMP8 in cross presentation is also shown by using VAMP8-/- cells.

SNAREs are important in vesicle transport and fusion. Therefore they can be targeted by pathogens like Leishmania to impair effective pathogen killing (Image from Wikipedia).

 

What is nice about this study is that by studying host-parasite interactions and immune modulation, it also helps us learn more about the innate immune mechanisms and communication of the innate and adaptive immune systems. 

 

Matheoud D, Moradin N, Bellemare-Pelletier A, Shio MT, Hong WJ, Olivier M, Gagnon E, Desjardins M, & Descoteaux A (2013). Leishmania Evades Host Immunity by Inhibiting Antigen Cross-Presentation through Direct Cleavage of the SNARE VAMP8. Cell host & microbe, 14 (1), 15-25 PMID: 23870310

Olivier M, Atayde VD, Isnard A, Hassani K, & Shio MT (2012). Leishmania virulence factors: focus on the metalloprotease GP63. Microbes and infection / Institut Pasteur, 14 (15), 1377-89 PMID: 22683718
 

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Finding new vaccine and diagnostic targets using Immunoproteomics

Posted by Kasra

One of the complexities in studying eukaryotic parasites is the multiplicity of their life stages. Even the simplest life cycles of eukaryotic parasites can consist of two very different stages, with different morphologies, gene expression, proteome profiles, and surface antigens. These variations often result in confusion of the immune system and disease progression instead of healing. What makes this more complicated is that very often one or more of these stages, usually the one inside the mammalian host can be difficult to culture and study in vitro. For instance, in the case of Leishmania parasites, the clinically important amastigote stage is intracellular. Methods for their axenic growth do exist; still their validity and authenticity remains controversial among researchers. Nevertheless, I believe authentic or not, axenic Leishmania amastigotes can be good tools for studying this aloof life stage of the parasite. As the famous statistician George P.E. Box says ‘Essentially, all models are wrong, but some of them are useful’.

Yet another complexity of working with these ancient species is presence of a great percentage of what genome annotators call ‘Hypothetical proteins’. These proteins appear after bioinformatic analyses of the genome sequences in search for genes. There is no other evidence rather than clues from the sequence for their existence, so they are labeled hypothetical. In addition, in many cases they have no homology to any protein with a known function, thus their function remains a big question mark, which brings me to the two papers I want to discuss!

These papers both came out last year and used immunoproteomics to hunt for new diagnostic and vaccine targets for leishmaniasis. Vinicio T. S. Coelho et al. ran 2D gels of promastigote and axenic amastigotes of Leishmania chagasi, a visceral leishmaniasis-causing species in Latin America, and blotted them against pooled sera of infected, uninfected or nonsympomatic dogs. Míriam M. Costa et al. used the colourful 2D-Difference Gel Electrophoresis (DIGE) method to look at differentially expressed proteins between promastigotes and amastigotes and also blotted them against pooled sera of uninfected, 30 day infected and chronically infected dogs to compare levels of early and late (IgM and IgG) antibodies. Both studies aimed to find immunogenic proteins as candidates for diagnosis and vaccination. For those who are not familiar with the ecology of Leishmania, I should mention that leishmaniasis is a zoonosis, and dogs are an important reservoir of the parasite that keep the cycle going, even if we prevent it in humans. Thus, vaccination of dogs against both cutaneous and visceral leishmaniasis is among the important priorities for disease control.

A combination of Difference Gel Electrophoresis and western blotting using sera allows for identification of common and stage-specific antigens. Míriam M. Costa et al. J. Proteome Research, 2011

The importance of these two studies is the application of both promastigote and amastigote proteins as sources for antigen discovery, as well as the use of sera from asymptomatic versus symptomatic dogs to characterize antibodies that arise at different stages of infection. This allows for identification of proper markers for early and advanced stages of the disease as well as knowledge about expression and antigenicity of proteins from each life stage of the parasite. Not surprisingly, in both studies, a decent number of hypothetical proteins show up. On one hand, these are not the best candidates one may look for, since we have no knowledge about their expression, function and so on. But on the other hand, I would see them as potentially interesting targets that could be worth studying. At least, we are narrowing down all the hypothetical proteins to ones for which we have data on expression and antigenicity.

In addition, the results of these studies and other studies of the similar nature should be cross-referenced in the public gene and protein databases, so that other researchers can readily access the new knowledge that has become available about these hypothetical proteins when looking them up. Once these sorts of data from various stydies start to accumulate in the databases, new patterns and insights might emerge that can lead us to an understanding of their function and possible roles in pathogenicity.

Coelho VT, Oliveira JS, Valadares DG, Chávez-Fumagalli MA, Duarte MC, Lage PS, Soto M, Santoro MM, Tavares CA, Fernandes AP, & Coelho EA (2012). Identification of Proteins in Promastigote and Amastigote-like Leishmania Using an Immunoproteomic Approach. PLoS neglected tropical diseases, 6 (1) PMID: 22272364

Costa MM, Andrade HM, Bartholomeu DC, Freitas LM, Pires SF, Chapeaurouge AD, Perales J, Ferreira AT, Giusta MS, Melo MN, & Gazzinelli RT (2011). Analysis of Leishmania chagasi by 2-D difference gel electrophoresis (2-D DIGE) and immunoproteomic: identification of novel candidate antigens for diagnostic tests and vaccine. Journal of proteome research, 10 (5), 2172-84 PMID: 21355625

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Parasites to help fight cancer

Posted by Kasra

Research on parasites is important, even if most of them are not direct health concerns to the developed world! Millions of years of coevolution of parasites along with their hosts have made them masters in manipulation of the immune system and in coexistence with it. Many parasitic infections such as those with Toxoplasma, Trypanosoma and some Leishmania sp. elicit innate and adaptive immune responses that can result in life-long immunity to reinfection. However, the parasite might be carried chronically for life, keeping the antibody titers up.

The research team of Ricardo Gazzinelli have taken advantage of the stealth yet stimulatory property of parasites to target cancer. For their purpose, they expressed a cancer antigen in a strongly attenuated strain of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. This live vaccine showed great protection against melanoma in both prophylactic and therapeutic models.

T. cruzi was chosen as the vector for many reasons: it intrinsically possesses TLR ligands and thus induces a strong proinflammatory response; like many other parasites, it has ways of staying inside the body for a very long time; and it propagates inside the cytoplasm, therefore it can induce a Th1 type immune response and activation of cytotoxic CD8+ T cells, which are very important against cancer. It is interesting to me that although they did not observe any disease or parasitemia, such strong immune response and protection was observed. I think it is important to see if and how many residual parasites are sticking around and where and how long do they stay in the body. This might also teach us more about how this model actually works.

The study shows that if vaccinated before tumor induction, mice are completely protected from cancer. It is also shown that this antigen-carrying vector can delay tumor growth and lethality if given after. One might ask: what advantages does this vector provide over vaccination with traditional recombinant protein, or other vectors such as attenuated viruses? The authors compared the protection and immune response resulted from recombinant T. cruzi to the canonical recombinant antigen (NY-ESO1) with Alum or CpG adjuvants. Recombinant T. cruzi showed better protection and stronger immune response compared to canonical vaccination strategies. Doing a quick search through pubmed, I didn’t find other strategies such live or viral vectors rather than variations of recombinant protein being used so far. I might be wrong, but still this is a novel idea and this recombinant parasite deserves a chance of being further studied, especially for prophylactic uses. There are plenty of other cancer or maybe even infectious antigens that can be targeted by this method. But of course, there are plenty of biohazard issues that also need to be addressed with injecting an attenuated form a dangerous pathogenic parasite.

Here is the link to the article abstract on Pubmed.

Junqueira C, Santos LI, Galvão-Filho B, Teixeira SM, Rodrigues FG, Darocha WD, Chiari E, Jungbluth AA, Ritter G, Gnjatic S, Old LJ, & Gazzinelli RT (2011). Trypanosoma cruzi as an effective cancer antigen delivery vector. Proceedings of the National Academy of Sciences of the United States of America, 108 (49), 19695-700 PMID: 22114198

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