Monday, September 30, 2013

HIV restriction factor- Mx2

Greetings

     More recently, I have been posting a lot on HIV. Someone commented, "Guess this blog has to be called as "Retro-virology" rather than Medical Microbiology. There has been so many papers on HIV that appeared recently, that has been so interesting to me at least, that I intended to follow up with. Probably am biased in my literature search, as these days am concentrating more on HIV papers!!! Either way, its interesting enough.

     Viruses (irrespective of type) and the host cells have been in a constant competitive turn up against each other. Each trying to get the better of other. That's the "Red queen hypothesis". So, at least by virtue of evolution, we have selected for genetic factors and machinery that are dedicated to cellular immunity. So, it wouldn't come as a surprise, if I told you that the the cells have some discovered (and possibly many undiscovered), factors that act as HIV restriction factors.

      Well studied HIV restriction factors include- Cyclophilin A, APOBEC3, TRIM-5α, SAMHD1 and BST-2. The details of these has been shown in Table 1.
Table 1: Major HIV restriction factors.
     MX2 (Myxovirus Resistance 2) protein coded by MX2 gene, is a type of Dyanamin like GTPase occuring in 2 forms- Nuclear form and cytoplasmic form. The nuclear form has a NLS (nuclear localisation signal, whereas cytoplasmic form doesn't (NLS is present at the amino terminal end of the nuclear form but not in cytoplasmic form due to use of an alternate translation start codon). The Mx proteins are implicated in the interferon type I signalling mediated innate antiviral response. Human MxA GTPase known to be localised with intracellular membranes, preferentially in Endoplasmic Reticulum- Golgi intermediate compartment, which is used by many viral pathogens as an intracellular replication site.

Fig 1: Proposed mechanism of
Mx functioning.
Source
    The crystal structure of MXa protein studied by Gao etal, suggested Mx proteins contains 3 domains-

1. Amino-terminal (N-terminal) GTPase (G) domain
2. Central middle domain (MD)
3. Carboxy-terminal (C-terminal) GTPase effector domain (GED)

    It is proposed that the antiviral activity of MxA would be dependent
on GTP hydrolysis performed by the G domain and oligomerization mediated by the MD and GED. Current understanding speculates that the Mx possibly interacts with viral ribonucleoprotein, and thus inhibiting the replication. See Fig 1. The structure and function of other Mx proteins is not expected to differ much.

   A study published in Nature by Goujon etal, has showed that this factor is probably important in HIV infection, as a restriction factor. This is based on the observation that, cell lines where Mx2 was switched off, showed better replication in comparison to the cells where Mx2 is on. However, the surprise is that Mx1 (Discussed above), cannot curb the HIV. Mx2 is effective against HIV not Influenza and Mx1 is vice versa.

     This has important implications. As the authors suggest- there can be 2 therapeutic approaches designed based on this concept. First, we can make an analogue of Mx2 that can serve the same function or develop a compound that can increase the activity of Mx2 protein.

ResearchBlogging.orgGao S, von der Malsburg A, Dick A, Faelber K, Schröder GF, Haller O, Kochs G, & Daumke O (2011). Structure of myxovirus resistance protein a reveals intra- and intermolecular domain interactions required for the antiviral function. Immunity, 35 (4), 514-25 PMID:21962493

Goujon C, Moncorgé O, Bauby H, Doyle T, Ward CC, Schaller T, Hué S, Barclay WS, Schulz R, & Malim MH (2013). Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection. Nature PMID:24048477

Sunday, September 29, 2013

HIV Toolbox

Greetings

    In my last 2 blog posts, I have been talking about how hard the researchers are trying to develop a vaccine (includes both humoral and cellular immunity based vaccines). Though many approaches have failed to demonstrate a high level protection, and the scientists have reached a point of frustration, we have reached a time scale where, we are on the verge of development of novel strategies. HIV is probably one of those pathogen, that has defied the science, with the challenge still up in the air for anyone willing to catch it.

Fig 1: HIV intervention
   HIV-1 is intervened by several different approaches (Often referred vaguely in literature as the HIV toolbox). This has been an ever expanding list and there has been a great deal of progress in the existing category. The list shown in Fig 1, is by no means a complete data but, at least on the day of writing serve a backbone.

    In the last couple of weeks there has been some remarkable papers in this field, and i just want to put a very brief summary of these, as most of the ideas pertaining to these, I have discussed somewhere in my previous posts.

      bnAb (Broadly neutralizing Antibodies), has come up as a great deal of interest, especially in case of HIV and Influenza, where there is too much of antigenic variation, to attack using a single type of antibody. One of the well studied antibody is 4E10, is probably the neutralizing antibody with maximum spectrum binding known, recognizing a highly conserved epitope in the membrane-proximal region of gp41. Another well studied is b12 which overlaps the CD4 receptor-binding site (CD4bs) on gp120. Now the researchers from The Scripps Research Institute (TSRI) have shown that the 4E10, which has been of great promise, is also reactive against self antigens (Probably through cross reaction), whereas b12 is not. David Nemazee, a senior author said. "It's still possible that we could safely elicit the 4E10-like antibody in order to protect against HIV, We just have to think about how to generate the best antibodies without causing other problems. We have a lot of questions. And now we have a good model to help us answer them". Source

Fig 2: Ciclopirox
Source
      A yet another study has to do with, serendipitous observation that ciclopirox, used as an anti- fungal for treating skin infections can also attack HIV. The study looked at the ability of 2 well used pharmacological drugs- topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. HIV delays or blocks the cellular apoptotic program, which seems to be reactivated in this case. The evidence comes form the activation markers- caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology of infected cells. However, the best part of this study is that there was no viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir.

Fig 3: DAVEI activity
Source
     The third paper that was published earlier, is based on a lab construct microbicide referred as "DAVEI" (Dual Action Virolytic Entry Inhibitor). This is a chimeric recombinant protein, constructed of a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)x linker (where x is 4 or 8) between the C terminus of the former and N terminus of the latter. Its long been testified based on earlier studies that the HIV fusion machinery is based on the idea of Spring load mechanism (Thats the basis of fusion inhibitors). The MPER component is is itself a small piece of the fusion machinery and interacts strongly with viral membranes. Cyanovirin, binds to the sugar coating of the protein spike. The binding of virus, gets the virus to be signaled that it has attached to a cell, and hence releases genetic material, where in reality there is no cell. This leaks the genes out, which is harmless. Prrr.. Tricked the virus!!!   Chaiken said "DAVEI and other new-generation virolytic inactivators open up an important opportunity to develop a topical microbicide to block the transmission of HIV, and at the same time provide lead ideas to discover treatment strategies for people who are already infected, Our hope is that determining the structural driving forces of both inhibitors and viral entry machinery that enable spike inactivation will help to advance molecular designs with increased power, specificity and clinical potential for both prevention and treatment" Source

ResearchBlogging.org
Doyle-Cooper C, Hudson KE, Cooper AB, Ota T, Skog P, Dawson PE, Zwick MB, Schief WR, Burton DR, & Nemazee D (2013). Immune Tolerance Negatively Regulates B Cells in Knock-In Mice Expressing Broadly Neutralizing HIV Antibody 4E10. Journal of immunology (Baltimore, Md. : 1950), 191 (6), 3186-91 PMID: 23940276

Hanauske-Abel, Hartmut M. (2013-09-23) Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection. , 8(9), e74414. DOI: 10.1371/journal.pone.0074414

Contarino M, Bastian AR, Kalyana Sundaram RV, McFadden K, Duffy C, Gangupomu V, Baker M, Abrams C, & Chaiken I (2013). Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1. Antimicrobial agents and chemotherapy, 57 (10), 4743-50 PMID: 23856780

Friday, September 20, 2013

Critical analysis of HIV vaccines- Part II

Greetings

     Yesterday, I had posted about the basic facts of what makes an HIV vaccine difficult, The AIDSVAX and STEP trial which failed. I also talked about the possible reasons of why the vaccine probably failed, and what research is on way to overcome the problem. Continuing from my earlier post, here I talk about alternative options under development that may interest the researchers.

Fig 1: Env Ab negative correlation with HIV viremia
    Gag and Env are good immunogens, there maybe a good way to target them. Studies showing correlation between the antibodies and viral loads have been published. What comes as a surprise is that Gag antibody levels are correlated with reduced viral load, and Env-Ab levels with Increased viral load. I have not found an explanation for this phenomenon. The study done in a small population, has brought in the question of is Env antigen a candidate for vaccine? Moreover, studies have shown that there is a strong association between gag and env evolution, and env sequence diversion is higher in chronic patients. Of course, every single variation will not make an appearance, for obvious reasons and a natural molecular selection plays significant role. However, even a 1% increase in divergence is significantly linked to higher viral load, as the immune system has to re calibrate the response.

Photo 1: Intradermal Electroporation
Applicator. Source
      Am highly tempted to argue that possibly Gag represents a good candidate, atleast one which can direct useful antibodies. A very recent publication uses a DNA vaccine approach. Programmed death-1 (PD1) linked to HIV-1 GAG p24 antigen (Fusion DNA product), delivered by intramuscular immunization via electroporation (EP), in mice elicited consistently high frequencies of GAG-specific, broadly reactive, polyfunctional, long-lived, and cytotoxic CD8+ T cells and robust anti-GAG antibody titers. Though Electroporation is not a good option for human use, this maybe tweaked using vector delivery system, and hence is a possible approach. A yet another study by Inovio Pharmaceuticals (Link), where similar approach was conducted using DNA plasmids targeting the gag, pol, and env proteins of HIV-1, delivered by electroporation (CELLECTRA® electroporation device) . In a announcement reported on Mar 13, 2012, the study claimed that the study had achieved a strong T cell immune responses in a Phase I clinical study targeting treatment of the HIV subtype prevalent in North America and Europe, in HIV-positive subjects.

       In my previous post, I had said that RV144 had achieved an efficacy of about 30% and represented an important breakthrough. The study has been further advanced as a follow up known as RV305. The study was conducted by MHRP (U.S. Military HIV Research Program) in Thailand to evaluate re-boosting in volunteers who participated in the RV144 study. The next step- RV306 was expected to be launched as soon as posible. It will be aimed at comparing additional vaccine boosts in 360 new volunteers. RV306 study will be conducted at three sites: the Vaccine Trial Centre at Mahidol University and the Royal Thai Army Armed Forces Research Institute of Medical Sciences (AFRIMS), both in Bangkok, and the Royal Institute for Health Sciences (RIHES) in Chiang Mai. Lisa Reilly, the MHRP’s communications director says, "It is not an efficacy study, so it does not need to be large… We are hoping to conduct an efficacy study with an improved vaccine boost/adjuvant in Thailand, but it will not start until 2016/17". Source

      That brings me to the last but the latest research in HIV vaccination. What is probably the most important factor that hampers the HIV vaccine efficacy? There are once again several set of answers in this area. Other than the fact that virus keep changing for immune system to keep track (Already discussed in previous post), HIV-1 cannot replicate in dendritic cells (DCs), and that HIV-1 replication in macrophages is not very efficient. This is sometime referred as lack of immune stimulation. HIV-2 can replicate well in these cells (Posses Vpx, which counteract inhibitory effect of SAMHD1 in these cells). Point is they are less pathogenic. So one of the hypothesis was if we could the immune system constantly could be surveilling for HIV maybe we can achieve non sterilizing immunity.

    Following up on the hypothesis, Picker etal, constructed a RhCMV ( rhesus cytomegalovirus vector), encoding the simian immunodeficiency virus (SIV) mac239 proteins Env, Pol, Gag, and Vpr/Vpx. This established persistent, high frequency, SIV-specific effector-memory T cell (TEM) responses at potential sites of SIV replication in rhesus macaques (RM) and stringently control highly pathogenic SIVmac239 infection early after mucosal challenge. The study compared 4 groups-

*Group A- 12 macaques were given the rhCMV/SIV viral vector-based vaccine
*Group B- 12 received an rhCMV/SIV vector-based candidate followed by a replication-defective adenovirus serotype 5 (Ad5) vector-based candidate encoding the full SIVmac239 genome
*Group C- 9 received a DNA prime/Ad5 boost
*Group D- 28 unvaccinated control animals

     In this setup, the Group C and D, exhibited typical progressive SIV infection. The Group A and B showed complete control of SIV. This study land- marked a proof of concept. In a recent follow up study published by the same group, showed that the vaccine suppresses SIV to undetectable levels in about 50% of the animals after vaginal and intravenous challenge as well. U can listen to the authors views on this study here.

     I want to call your attention to the STEP trial failure. The vaccine was awesomely successful in primate models. The failure in clinical trial has been attributed to Ad5 antibodies. Given the fact that CMV is also a common infection of humans, antibodies will be present against CMV. Will this cause the same effect as of STEP trial?? I couldn't find an answer to this case. Perhaps, I need to wait for some more data or I haven't grabbed the concept well.

     But one comment for sure. We have a long way to go

ResearchBlogging.org
Piantadosi A, Chohan B, Panteleeff D, Baeten JM, Mandaliya K, Ndinya-Achola JO, & Overbaugh J (2009). HIV-1 evolution in gag and env is highly correlated but exhibits different relationships with viral load and the immune response. AIDS (London, England), 23 (5), 579-87 PMID: 19516110

Zhou J, Cheung AK, Tan Z, Wang H, Yu W, Du Y, Kang Y, Lu X, Liu L, Yuen KY, & Chen Z (2013). PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice. The Journal of clinical investigation, 123 (6), 2629-42 PMID: 23635778

Hansen SG, Ford JC, Lewis MS, Ventura AB, Hughes CM, Coyne-Johnson L, Whizin N, Oswald K, Shoemaker R, Swanson T, Legasse AW, Chiuchiolo MJ, Parks CL, Axthelm MK, Nelson JA, Jarvis MA, Piatak M Jr, Lifson JD, & Picker LJ (2011). Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine. Nature, 473 (7348), 523-7 PMID: 21562493

Hansen SG, Jr MP, Ventura AB, Hughes CM, Gilbride RM, Ford JC, Oswald K, Shoemaker R, Li Y, Lewis MS, Gilliam AN, Xu G, Whizin N, Burwitz BJ, Planer SL, Turner JM, Legasse AW, Axthelm MK, Nelson JA, Früh K, Sacha JB, Estes JD, Keele BF, Edlefsen PT, Lifson JD, & Picker LJ (2013). Immune clearance of highly pathogenic SIV infection. Nature PMID: 24025770

Thursday, September 19, 2013

Critical analysis of HIV vaccines- Part I

Greetings

     If u have been a regular follower of my blog posts, more recently I have been posting often about vaccines, new vaccine strategies and topics revolving around that. When it comes to the vaccines, there is nothing of a more formidable challenger than the one compared to HIV vaccine. Millions of dollars spent, Years of research knowledge, and where do we stand? Thats a pretty good question to ask and is the topic of this post. When HIV was first discovered, Margaret Heckler declared "We hope to have such a vaccine ready for testing in approximately two years…yet another terrible disease is about to yield to patience, persistence and outright genius". That was in 1984. Even if I say this now, in 2013, I probably am going to be invariably wrong.

Fig 1: Neutralizing Antibodies cannot
access the constant region
      What makes the virus hard to be targeted by a vaccine? There are a couple of reason that can be suggested straight away. First, the virus is nastily variable at regions where antibody can reach. This is attributed a very sloppy reverse transcriptase, that induces a huge lot of variation. If all the different HIV from a single patient could be sequenced, we will note that there is still a plethora of variation. This makes it hard for the immune system to recognize and attack. The 2nd important problem is that the virus attacks the immune system itself- the CD4+ T cells that actually is the master mind of Immunological attack.

     One way to get around the problem of attacking the broad range of antigenic configuration is to take the path of Broadly neutralizing antibodies. (I have detailed the theory here). Any given immune response can be considered under 2 headings- Sterilizing immunity (Total wipe out of pathogen) or Non sterilizing immunity (Keeps the pathogen under surveillance and controls pathogen load). What is desirable is sterilizing immunity which requires Neutralization antibodies. However, if that isn't achievable we could still be contend with the other mode. 

    There have been an zillion candidates tried against HIV, of which 4 have made it to Phase III trials- AIDSVAX (2 versions), STEP trial and RV144 trial. VOICE (Vaginal and Oral Interventions to Control the Epidemic) Trial and CAPRISA 0004 (Centre for the AIDS Programme of Research in South Africa), are other important Clinical studies when it comes to HIV prevention. But they are microbicides based approach, a topic to be talked about separately.

AIDSVAX:

    This is a bivalent vaccine consisting of a preparation of recombinant gp120 from two types of HIV, is being developed by VaxGen for the potential prevention of HIV infection. Its earlier version was a monvalent vaccine. The vaccine was reportedly a failure, as per results published in 2003 (multiple papers). The results of AIDSVAX Trial shown below is least impressive. Developments are on way

Fig 2: AIDSVAX Trial results. Source

STEP trial (Phambili Trial):

Fig 3: Anti–HIV-1 T cell responses
Source
    The trial was based on the initial findings of MERCK laboratory that concentrated on the cellular arm of immunity. The rationale was to look for candidates that elicit higher CMI. Based on sampling from multiple clades (HIV-1 clades A, B, and C), each were compared by standardized interferon- gamma enzyme-linked immunospot assays among unvaccinated individuals, infected with diverse HIV-1 clades, from Brazil, Malawi, South Africa, Thailand, and the United States. As seen in the Fig 3, though there is a significant difference in immune response, Gag, Pol, Nef, and Env proteins are shown to be best targeted.

Fig 4: AD5 vaccine, developed by MERCK and NIH
Source
     Based on the findings said above, Merck developed the experimental vaccine called V520 to stimulate HIV-specific CMI. The vaccine was made from a replication incompetent Adenovirus vector 5 (Adv5) expressing Gag, Pol, Nef. The STEP trial was actually conducted in US and other countries and the Phambili trial was conducted in South Africa. However, now all the STEP trial is represented as the Phambili trial. Whatever is the case, the trial was halted owing to the observation that the vaccine group presented with higher infection rate than the placebo. That was a hefty un-expected blow. In article that appeared in NEJM (Link), Anthony Fauci, the director of the NIAID was quoted as "“To be brutally honest with ourselves, we have to leave open the possibility. . . that we might not ever get a vaccine for HIV. People are afraid to say that because they think it would then indicate that maybe we are giving up. We are not giving up. We are going to push this agenda as aggressively and energetically as we always have. But there is a possibility— a clear finite possibility — that that’s the case.”

      Natural scientific instinct was to look for cause. There were several proposed explanations as to what may be the reason for vaccine failure. The most noted explanation is as follows. In ana experiment where antibodies to adenovirus 5 was combined with the vaccine backbone (Ad5 Immune Complex ), they triggered more notable DC maturation, which increased CD86 expression, decreased endocytosis, and production of tumor necrosis factor and type I interferons. This led to activation of T cells, which are targeted by HIV in the body. In cell culture, T cells succumbed to HIV-1 infection three times more quickly than did those in a mixture that lacked adenovirus 5 antibodies. Since Ad5 is a very common virus and people may easily have antibodies this is a very plausible explanation. There are debates as to if this really is a case, but otherwise is accepted at least as a partial theory.

      That leads me to a question. If the Ad5 in itself is the problem, how about an alternative vector. The question seems to be very compelling, but answer is not easy to come. There are alternative adenovirus vectors under development such as Ad35 and Ad41. However, their toxicity and expression profiles are not much different. Another development is the use of modified recombinant Ad5 vector deleted for E1 and E2b deleted. In a study by Osada etal, it was shown that [E1-, E2b-]vectors retaining the Ad5 serotype are potent immunogens in pre-clinical models despite the presence of significant Ad5-specific immunity, in contrast to [E1-] vectors which was used earlier. Other vectors have been explored such as MVA and  NYVAC, which  are efficient in inducing both cellular and humoral responses against HIV-1.

Fig 5: Different HIV-1 vaccine strategies
Source
     I want to make a digression here. MERCK Ad5 vaccine was not the only vaccine that MERCK has tried or is under process. Other methods used to elicit CMI include- Plasmid DNA CRL 1005 formulation, Pox virus vector based vaccines (Vaccinia,  Modified vaccinia Ankara virus; MVA, Canary pox; ALVAC), BCG vector, Alphavirus vector (VEE), Virus Like particles (VLP) and and liposome delivery methods. Of these Plasmid DNA CRL 1005 formulation, Alvac are the ones under active investigation. 

RV144 (Thai HIV vaccine) trial
Fig 6: Efficacy of RV144, microbicides and PrEP
Source
  Alvac and AIDSVAX given in combination has been tried in this trial. This involved 4 priming injections of a recombinant canarypox vector vaccine (ALVAC-HIV [vCP1521]) plus two booster injections of a recombinant glycoprotein 120 subunit vaccine (AIDSVAX B/E). The maximum vaccine efficacy was approx about 30%. (Phew!!! After 6 total doses, just 30%?). Experts have argued on both grounds. One group says 30% is not good enough. There are other people who comment, 30% is a good starting point, given the fact that the most promising STEP trial showed negative efficacy. Often the outcome of this trial is compared with the microbicides, as shown in Fig 6.

     I will continue regarding our current understanding and breakthroughs in this field, in the next post.

ResearchBlogging.org
Coplan PM, Gupta SB, Dubey SA, Pitisuttithum P, Nikas A, Mbewe B, Vardas E, Schechter M, Kallas EG, Freed DC, Fu TM, Mast CT, Puthavathana P, Kublin J, Brown Collins K, Chisi J, Pendame R, Thaler SJ, Gray G, Mcintyre J, Straus WL, Condra JH, Mehrotra DV, Guess HA, Emini EA, & Shiver JW (2005). Cross-reactivity of anti-HIV-1 T cell immune responses among the major HIV-1 clades in HIV-1-positive individuals from 4 continents. The Journal of infectious diseases, 191 (9), 1427-34 PMID: 15809900

Steinbrook R (2007). One step forward, two steps back--will there ever be an AIDS vaccine? The New England journal of medicine, 357 (26), 2653-5 PMID: 18160684

Perreau, M. (2008-11-03) Activation of a dendritic cell-T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells. , 205(12), 2717-2725. PMID: 18981239

Osada T, Yang XY, Hartman ZC, Glass O, Hodges BL, Niedzwiecki D, Morse MA, Lyerly HK, Amalfitano A, & Clay TM (2009). Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing anti-vector immunity. Cancer gene therapy, 16 (9), 673-82 PMID: 19229288

Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de Souza M, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH, & MOPH-TAVEG Investigators (2009). Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. The New England journal of medicine, 361 (23), 2209-20 PMID: 19843557

Thursday, September 12, 2013

Headlines- MERS and MERS vaccine

Greetings,

     In my previous blog-post, I put up about the publication of a potential vaccine candidate against influenza, which can be made in almost a weeks notice. Influenza is a problem that pops up every 3-4 years, with a new strain gaining world wide scientific attention. Probably this is the first time a novel coronavirus (nCoV),   now called as MERS (Middle East respiratory syndrome coronavirus) gained almost equivalent popularity as that of Influenza. Clearly lot of research is focussed on this problem. Once again a vaccine candidate has grabbed my attention, and so this post.

Fig 1: CoV classification.
Source
   Coronaviruses (CoV; member of nidovirales), are so named for their appearance. The virus is enveloped, genetic material containing a single stranded RNA (+ sense) and a helical symmetry nucleocapsid. Human coronavirus infections with majority of them presenting as asymptomatic respiratory infection, and rarely a few causing moderate to severe illness. The coronaviruses are sub-classified into alpha (Group I), beta (Group II), gamma (Group III) , and delta (Group IV) coronaviruses. The most famous of the coronavirus is SARS (Severe acute respiratory syndrome- CoV). Other well studied human pathogens include HCoV-229E and HCoV-OC43.

    MERS coronavirus, previously referred as Novel CoV (nCoV), is a lineage C member of the Beta- coronavirus group. The first case was seen on 31st May 2012, laboratory confirmed in Italy. The Index case was a previously healthy man, who had returned to Florence following a 40-day holiday in Jordan. He had signs of pneumonia, with bilateral lung infiltrates. In the following days, two more cases appeared, from people in close contact with the index case. The laboratory confirmation was based on a Real-Time PCR for genome upstream of the E gene (upE region). This was later also confirmed by National Influenza Center. There has been regular reporting of MERS CoV infection since. A week ago, WHO recorded a total of 114 global cases, of which 54 expired. On the date of this blog being posted, 16 more have been reported by Saudi Arabia's health ministry, making it to a total of 130 cases, of which 57 have been fatal (Update from CIDRAP; Link).

Photo 1: Taphozous perforatus
Egyptian tomb bat
      A great deal of work is currently in process, as to identification of the source. A wide range of serological test for detecting sero-conversion in animals has been done. Antibodies have been detected against the spike of MERS and has lead researchers to conclude that MERS has at some point of time, passed into camels. A study by Lipkin's group showed that they could isolate a small sequence(190 nt sequence, with maximum possibility of identity) in a faecal sample from an Egyptian tomb bat. They identified only small sequence just once. However, the press nailed it as "confirmed transmission", which is not agreed upon by the author itself (Listen to an interview with Lipkin in TWiV 247; Link). A quote from author “Although this fragment means a very close relative of the human MERS-CoV is found in a bat geographically close to the first case, the fact it is identical in this short region doesn’t mean that these bats are the direct source of the human case.” Source. Other comments include “It can absolutely not be ruled out that it is a sequence derived not from MERS-CoV but from another, closely related MERS-CoV like virus”. and "The finding is, of course, important, but it has to be reproduced by others, and it has to be found in other bats.” Source

Fig 2: Life cycle of MERS-CoV.
Source
     The basic biology of CoV is very much similar to SARS CoV. Most of the studies done and mechanisms that has been worked out, has been guided from findings of SARS virus. From the DNA sequences that have been studied, it has been estimated that there are 10 open reading frames, expressed through nested set of 8 mRNAs.

   Of all, the most important finding was the cellular receptor- dipeptidyl peptidase 4 (DPP4). The DPP4 binds to a 231-residue region in the spike (S) protein of MERS-CoV. The RNA genome is pumped in through a plasma or endosomal membrane fusion, into the target cell. The RNA immediately transcribes to proteins and RNA, which is packaged and released. See Fig 2.

    As you would gather from the above, the number of reported cases isn't huge of a deal. But what has been the issue is case fatality rate. The virus is now slowly opening up to a spreading potential. The best thing that a pathogen can have is ability to spread well, which can be well inferred from the current trend. Clearly we need a vaccine.

     When I say vaccine, the first thing that I can think about is antibodies against Spike protein. A genetically stable modified strain Vaccinia Virus Ankara (MVA) expressing full-length spike protein (MVA-MERS-S) has been constructed that induces neutralizing antibodies in mice model. I don't know much of the details about this as I haven't read the paper. Sorry, but I don't have access. But I can vaguely assume that it is a sensible approach. Probably, we could also try sub- component vaccine Just the Spike protein expressing only the "231-residue binding region" would have any improved effect? (Am assuming that just the epitope would be more specific. I have no idea if that would have any alternate or better results).
 
Fig 3: Growth kinetics of the deletion mutants.
    But that hasn't somehow made the headlines. But what did make a news is an engineered strain lacking the E gene (rMERS-CoV-E) that can replicate, but not propagate. The research story reads so. Mutations created in  3, 4a, 4b and 5 (accessory genes), had no significant effect on MERS- CoV replication and showed similar growth rates as that of wild- type virus. See Fig 3 taken from paper. But when the E gene was mutated there was a clear disability. The virus was replication competent but propagation defective. This is mostly due to requirement of E to mature.

     A question that arises here for me is "Does deleting E defeat the whole purpose of live attenuated vaccine?". The bang point of Live vaccines are that they are self replicating and hence doesn't need to be given multiple antigen doses. But since loss of E essentially cripples MERS virus and dies of quickly after producing some antigens. (Hmmm!!! Probably, I can give some more thought into this). Oh wait. So how did the researchers grow this strain then? Ah, simple. They integrated E gene into host chromosome (Which is not borrowed by virus), and so these "packaging cells", will support growth. However the new grown virus still doesn't possess E gene.

Photo 2: Camel
     Of course, we wont have a vaccine say in about next month. There is this triple hurdle rule (make at least 3 mutations in genes to avoid immediate recombinant revertants) to consider the use of genetically modified live attenuated vaccine for commercial clinical use. So it comes as no surprise to me when Dr. Peter Hotez comments, "There's a long way to go from showing in a research laboratory that you've got a potential candidate vaccine to actually producing a bottle of that vaccine that is going to be used or stockpiled". The same source says, an animal oriented vaccine (Maybe camels are the best candidate to start in the first place), maybe manufactured soon and deployed into the field. Source.

    But if camels are supposed to be mediating the spread is it appropriate to vaccinate them without the triple gene defect protection? I mean it could revert in animal and pass onto humans. I leave the debate to the reader.

ResearchBlogging.org
Puzelli S etal (2013). Investigation of an imported case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection in Florence, Italy, May to June 2013. Euro surveillance 18 (34) PMID:23987829

de Groot, R. J. (2013-07-15) Middle East Respiratory Syndrome Coronavirus (MERS-CoV): Announcement of the Coronavirus Study Group.J Virol. , 87(14), 7790-7792. PMID:23678167

Reusken CB etal (2013). Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. The Lancet infectious diseases PMID:23933067

Memish, Ziad A. (2013-11--1) Middle East Respiratory Syndrome Coronavirus in Bats, Saudi Arabia. EID , 19(11). DOI:10.3201/eid1911.131172

Raj VS, Mou H, Smits SL, Dekkers DH, Müller MA, Dijkman R, Muth D, Demmers JA, Zaki A, Fouchier RA, Thiel V, Drosten C, Rottier PJ, Osterhaus AD, Bosch BJ, & Haagmans BL (2013). Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature, 495 (7440), 251-4 PMID: 23486063

Lu L, Liu Q, Du L, & Jiang S (2013). Middle East respiratory syndrome coronavirus (MERS-CoV): challenges in identifying its source and controlling its spread. Microbes and infection / Institut Pasteur, 15 (8-9), 625-9 PMID: 23791956

Song F, Fux R, Provacia LB, Volz A, Eickmann M, Becker S, Osterhaus AD, Haagmans BL, & Sutter G (2013). Middle East Respiratory Syndrome Coronavirus (MERS-CoV) spike protein delivered by Modified Vaccinia Virus Ankara (MVA) efficiently induces virus-neutralizing antibodies. Journal of virology. PMID: 23986586

Almazán F, Dediego ML, Sola I, Zuñiga S, Nieto-Torres JL, Marquez-Jurado S, Andrés G, & Enjuanes L (2013). Engineering a replication-competent, propagation-defective middle East respiratory syndrome coronavirus as a vaccine candidate. mBio, 4 (5) PMID: 24023385

Wednesday, September 04, 2013

SAM vaccine for H7N9

Greetings,

Fig 1: H7N9 cases to 16 April 2013 (red circles),
and population densities of humans. Nature news
      Influenza, continues to be a serious issue. With emergence of a new pathogenic strain- H7N9 in china (Read previous post here and here), influenza research focussed on making a vaccine. Virtually nothing much is known about this strain except for its mutation that has conferred a gain of function. On 23 April, China's state news agency reported a 36-year-old man in serious condition in the city of Zaozhuang. The rest is history. The H7N9, is a deep reflection of how less we know of the influenza virus.

   A little bit of digression, related to H7N9 studies. If you remember the story of influenza ferret experiments (Link) that dramatically unfolded over time (Link), it has been clear that gain of function (GOF) experiments is under high scrutiny by the public, for most of them don't understand what actually is the nature of experiment. The issue scaled up to a global news with intervention of Federal agencies. Though the paper was finally published and made available in open access format, the trouble of publishing had probably got scientists thinking along. So when a group of scientists planned to do some similar GOF experiment, the public and science is on a debate with opinions broadcasted or publicly posted. This was deliberately done to allow open discussion, so as to avoid future interference problems (Probably!!).

     Influenza vaccine is a subject of huge research. New Influenza strains of interest arise with un-predicted antigenic combinations, which delays the vaccine production. Rapid production technologies such as the RNA vaccine approach (CureVac’s RNActive ® technology; Link), attempts for universal vaccine, cell-free gene assembly technique, DNA vaccines etc have gained a huge interest. So when the Nature published SAM vaccine for H7N9, I had to blog about it.

Fig 2: Self amplifying mRNA derived from Alphavirus
    This research is very much based on a technology referred as self-amplifying mRNA (SAM™) platform for rapid synthesis of vaccines. Technique is based on self amplifying RNA delivery, using lipid nanoparticles (LNP). Several papers have been published were a non amplifying RNA (In this case siRNA) delivered to cells through LNP was used to treat allergy, neoplasms etc. The basic structure of a SAM is shown in Fig 2. The gene of interest (Whatever is the protein wish to be coded for) can be inserted, bingo. In a paper by Geall etal, a proof of concept experiment was shown to be highly successful. The experiment used self-amplifying RNA vaccines called stable nucleic acid lipid particles, delivered using DLinDMA (ionizable cationic lipid 1,2-dilinoleyloxy- 3-dimethylaminopropane).

    HA gene was synthesized from the A/Shanghai/2/2013, subsequently cloned into SAM vaccine genetic control elements and a T7 polymerase promoter. The copy was transcribed and transfected to BHK cell lines, checked for integrity and expression patterns. Finally, it was packaged into the LNP delivery system (Now referred completely as SAM/LNP), and tested against mice. The total time taken from identification of sequence to making of SAM, just 8 days (Refer Fig 3). Thats the fastest I have ever heard. By the way they tested its activity against mice and showed a good quality response, with 2 doses. Quote from article "With further streamlining, it should be possible to produce similar vaccine candidates within 5 days of the gene sequence of the virus being available".

Fig 3: Timeline of SAM H7N9 vaccine production. Source
  As the article points out, this methodology has several advantages. The most important is use of cell free system to synthesize the HA gene (nucleic acid amplification technologies such as PCR. or isothermal techniques), and use of SAM technology to create vaccine with speed and accuracy. The only disadvantage is  the RNA half life, which requires some care.

   Take home message, if we are not prepared to battle when the virus appears, we can at the fastest possible time, with SAM technology.

References:
   
ResearchBlogging.org
Malakoff D (2013). Avian influenza. Critics skeptical as flu scientists argue for controversial H7N9 studies. Science (New York, N.Y.), 341 (6146) PMID: 23926188

Jaffe HW, Patterson AP, & Lurie N (2013). Avian flu: Extra oversight for H7N9 experiments. Nature, 500 (7461) PMID: 23925232

Dormitzer PR etal (2013). Synthetic generation of influenza vaccine viruses for rapid response to pandemics. Science translational medicine, 5 (185) PMID: 23677594

Geall AJ etal (2012). Nonviral delivery of self-amplifying RNA vaccines. PNAS, 109 (36), 14604-9 PMID: 22908294

Armin Hekele etal (2013). Rapidly produced SAMH vaccine against H7N9 influenza is immunogenic in mice. Emerging Microbes and Infections 2, e52. doi:10.1038/emi.2013.54

Monday, September 02, 2013

No Tit for HIV TAT

Greetings

Fig 1: Structure of HIV TAT
Source
     In my previous blog post, I had put up our current understanding of HIV latency. Of course that was a very short post, and by no means, represent the complete picture of what we know. But, it did spill the idea in the right place. As I have pointed out in my previous post, TAT (Trans-Activator of Transcription), is essentially involved in regulating the latency and HIV genome expression. Its a common knowledge that HIV TAT is expressed in high quantities in circulation. It is almost evident from current research that the TAT contributes to additional pathology of HIV, especially in Neuro-AIDS. That is interesting enough to be worth a discussion here.

Fig 2: TAT toxicity
     HIV TAT is a central molecule of HIV (If I may say so). Once a proviral DNA is integrated into the host genome, the expression initially is controlled by a promoter sequence that can be activated by NF-kB. However, this usually is not enough, as it doesn't produce complete RNA (Owing to multiple blocks). Short transcripts produced during the course, allows making of TAT, that along with TAR (trans-activating response element), removes the block thereby allowing complete transcription. This forms the central cellular circuit in deciding HIV genetic expression.

Fig 3: Effects of TAT
        The second role of HIV TAT is to help in increasing HIV infection itself. How? Studies indicate that the HIV TAT is secreted in detectable quantities. The TAT uptake is by receptor mediated endocytosis using heparan sulfate (HS) proteoglycans, CD26 or LDL (low-density lipoprotein) receptor - related proteins. The TAT subsequently induces  transcription which leads to production of IL-2, IL-6 Tumor necrosis factor, CXCR4 and CCR5 cytokine co-receptors, Angiongenic factors etc. This increases inflammation, and simultaneously brings in new susceptible cells to the scene. Perhaps of all the effects that  in extracellular environment, the most important is its role in Neuro-AIDS (Especially its possible roles in AIDS dementia complex (ADC).


Fig 4: TAT based Neurotoxicity
     The mechanism of neurotoxicity of TAT is not well understood, is a subject of intense research in the field of Neurovirology. The current idea is that TAT effects, Neurons, astrocytes and Monocytes in CNS leading to extensive damage. Studies on human fetal neurons with TAT showed that the damage is independent of Na+ and preliminary studies suggested that TAT is possibly directly involved. The most widely accepted mode of action is speculated to be through production of reactive NO species (See Fig 4). TAT is also know to activate apoptosis in neuronal cells via activation of glutamate receptors of the non-N-methyl-D-aspartate. In the case of astrocytes, the same NO based damage can be initiated via interaction through CCL2 receptors. TAT can also directly up- regulate TNF-α dependent MCP-1/CCL2 production via MEK/ERK pathway. As mentioned earlier, the Endosome based uptake is the chief mechanism of TAT uptake in cells. Hence it is reasonable to find that the TAT toxicity is also mediated through interfering with the endolysosomal functioning. This property has been attributed to arginine-rich domain of HIV-1 Tat between amino acid residues 49 and 57.

     The above will leave an impression that TAT is a highly potential candidate to be attacked. There are 2 approaches that we can think of here. The first ofcourse is a vaccine design the other being pharmacological intervention. TAT antibodies are demonstrable in populations infected with HIV. The antibody response has been demonstrated in the vaccines- (i) TAT dominant B-cell epitope vaccine, using CyaA vector, which targets dendritic cells (ii) TUTI-16 is a synthetic universal HIV-1 Tat epitope vaccine. TAT inhibitors such as didehydro- Cortistatin A; dCA (derivative of Cortistatin A) has been shown to have significant activity. Of these, however to date there is no clinically approved therapeutic.

    Safe to say, we don't have a good theraupeutic against HIV TAT, which can significantly help the HIV patients.

ResearchBlogging.orgTyagi M, Rusnati M, Presta M, & Giacca M (2001). Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. The Journal of biological chemistry, 276 (5), 3254-61 PMID: 11024024

Hazleton JE, Berman JW, & Eugenin EA (2012). Purinergic receptors are required for HIV-1 infection of primary human macrophages. Journal of immunology (Baltimore, Md. : 1950), 188 (9), 4488-95 PMID: 22450808

Séror C, Melki MT, Subra F, Raza SQ, Bras M, Saïdi H, Nardacci R, Voisin L, Paoletti A, Law F, Martins I, Amendola A, Abdul-Sater AA, Ciccosanti F, Delelis O, Niedergang F, Thierry S, Said-Sadier N, Lamaze C, Métivier D, Estaquier J, Fimia GM, Falasca L, Casetti R, Modjtahedi N, Kanellopoulos J, Mouscadet JF, Ojcius DM, Piacentini M, Gougeon ML, Kroemer G, & Perfettini JL (2011). Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection. The Journal of experimental medicine, 208 (9), 1823-34 PMID: 21859844

King JE, Eugenin EA, Buckner CM, Berman JW. HIV tat and neurotoxicity. Microbes Infect. 2006 Apr; 8(5): 1347-57. PMID: 16697675

Magnuson DS, Knudsen BE, Geiger JD, Brownstone RM, & Nath A (1995). Human immunodeficiency virus type 1 tat activates non-N-methyl-D-aspartate excitatory amino acid receptors and causes neurotoxicity. Annals of neurology, 37 (3), 373-80. PMID: 7695237

Hui L, Chen X, Haughey NJ, & Geiger JD (2012). Role of endolysosomes in HIV-1 Tat-induced neurotoxicity. ASN neuro, 4 (4), 243-52 PMID: 22591512

Fayolle C, Davi M, Dong H, Ritzel D, Le Page A, Knipping F, Majlessi L, Ladant D, & Leclerc C (2010). Induction of anti-Tat neutralizing antibodies by the CyaA vector targeting dendritic cells: influence of the insertion site and of the delivery of multicopies of the dominant Tat B-cell epitope. Vaccine, 28 (42), 6930-41. PMID: 20728521

D'Orso I, Grunwell JR, Nakamura RL, Das C, & Frankel AD (2008). Targeting tat inhibitors in the assembly of human immunodeficiency virus type 1 transcription complexes. Journal of virology, 82 (19), 9492-504. PMID: 18667497

Guillaume Mousseau etal. Potent suppression of HIV viral replication by a novel inhibitor of Tat. Retrovirology. 2012; 9 (Suppl 1): O11.  PMCID: PMC3360218