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Valeura Energy OTC PNWRF TSX VLE
Patience finally paid off on this one as it did little for years.
Stock is up over 1000% since mid-Oct due to first deep well initial test results by Partner Statoil. It appears they have found a large BCGA in gas starved Turkey who has almost no in-country gas production and pays about triple the price paid in North America. Their Gas imports increased 25% in Nov. The first resource assessment for the basin is due out early Feb.
Here is a good summary of current outlook. If you look on the right side, you see his archives. He is the only reason I stuck it out.
http://hydracapital.ca/hydra-blog.html
JAN
11
Sorrento Therapeutics (SRNE) - Part 1 Brief Thoughts & Introduction
The BioWatch team recently traveled to the headquarters for Sorrento Therapeutics (SRNE) and met with CEO Henry Ji, VP of Immunotherapy Gunnar Kaufmann, as well as a number of scientists. Ji and his team graciously provided an extended Q&A as well as a general company tour. In evaluating SRNE, you should download the latest company presentation. It contains clinical data referenced in announcements and with their presentation at the JP Morgan Healthcare conference on January 11, 2016.
We first visited Sorrento in 2013. It was a much smaller, different company. We have witnessed its continuing evolution. It was fast. For the investor community, it’s even more jarring.
Sorrento now sports an unbelievably large, cutting-edge biotechnology portfolio. It has a portfolio that dwarfs any other “Small Bio”…and perhaps many “Pharmas” too. The investor community has this main question: Is it real or is it smoke and mirrors?
This 4-part update presents our impressions regarding Sorrento’s ability to execute on its portfolio. We will also discuss some of its key products. We won’t go into detail into all of its partnerships, at least not yet. We’ll also defer discussion of the CAR.TNK technology for our article on NantKwest – Sorrento’s “partner”. Unlike Sorrento, NantKwest is focused on one thing: Natural Killer cells.
Key Technologies & Products
Antibody Technologies
Cellular Therapies
Other
GMAB Library
Allogenic CAR.TNK
(Directed NK Cells)
Resiniferatoxin (RTX)
(Pain)
K-Lock & C-Lock Conjugation
CAR-T
(Directed T-Cells)
Cynviloq
(Licensed to NantPharma)
Mabtech’s BioSimilar and “BioBetter” mAbs
LA Cell Intracellular Targeting Technology
Bi-specific Antibodies
Various Stem Cell Targets
Lee’s Pharma (anti-PD-L1 mAb)
Furthermore, this update will provide notes to three Sorrento technologies:
1) “BioSimilar and BioBetter mAbs;
2) The intracellular targeting technology from the City of Hope (i.e. LA Cell);
3) Sorrento’s CAR-T platform.
Of the current drugs in development, we believe that the BioBetters will be the first late stage candidates that will bring significant value to SRNE, while the CAR-T and intracellular technologies are rarely discussed. The intracellular technology is real and rare.
Our Brief Thoughts
Sorrento has an extraordinary set of antibody technologies and cell therapies. It has an almost unbelievable number of shots on goal with each of its various platform technologies.
Is Sorrento real? Or is it smoke and mirrors?
Even if it’s real, SRNE will require time to percolate. It is still early. The antibody technologies and CART.TNKs are years away from reaching the market. This means that SRNE’s shorter-term valuation will be an upcoming reaction to milestones reached by its lead drugs, and any new partnerships.
Short-Term Milestones (Under 1 Year)
Sorrento doesn’t need to raise capital in the short-term, and hence it doesn’t attract significant investment banking attention. Its cash position appears healthy, and there’s more coming.
In May 15, 2015, NantPharma acquired Cynviloq from Sorrento. Whenever advances Cynviloq forward, then large regulatory payments will be directed towards Sorrento.
Sorrento also has Resiniferatoxin (RTX) for dealing with severe cancer pain. It should launch into late trials in 2016.
BioBetter mAbs
These are well-tested versions of approved and well-known mAbs in the USA. Sorrento acquired the rights to 4 biosimilar mAbs from Taizhou Mabtech Pharma in China. Late stage testing has already been completed in China. Sorrrento has exclusive rights to North America, Europe, and Japan.
Mabtech’s “BioBetters” originated from CHO cell lines while most of the “originals” were derived from recombinant yeast. Sorrento’s biosimilar mAbs thus have a better safety profile. This is why they’re called “BioBetters”.
We should see these mAbs enter Phase II/III trials in 2H 2016. While we hope these will become registration trials, it’s still a short path if an additional trial is still required.
Financial Update
(SRNE) recently filed its 10-Q for Q3 2015. We have provided selected data for summarizing its financial position. Sorrento has enough cash to last until mid-2016.
Basic Facts for Quarter Ending September 30, 2015
(in Thousands)
Q3-2015
Q2-2015
Q3-2014
Grants & Services Revenue
$ 1,103
$ 1,173
$ 1,276
Costs of Revenues
604
314
527
R&D Expense
7,244
7,971
5,440
Acquired in-process Rxh
24,068
-
-
G&A Expenses
4,711
3,072
1,854
Total Operating Expenses
36,738
11,706
8,407
Net Gain on Sale of Igdrasol**
69,274
-
-
Income Tax Expense
35,323
-
-
Net Loss
(2,079)
(10,958)
(7,605)
Basic & diluted loss per share
(0.03)
(0.30)
(0.27)
Avg. Shares Outstanding
37,328
36,315
28,533
Recent Price (per share)
7.81
(12/21/15)
21.17
(8/3/15)
8.51
(12/23/14)
Market Capitalization
295M
769M
243M
Cash & Marketable Securities*
$ 123,454
$ 51,699
$ 71,902
*SRNE owns about 5.6M shares of NantKwest NK
**Igdrasol held the IP and capital assets related to Cynviloq. It was sold to NantPharma
Sorrento doesn’t need to raise capital in the short-term, and hence it doesn’t attract significant investment banking attention. Furthermore, whenever NantPharma achieves regulatory milestones, then large payments will be directed towards Sorrento.
Any short-term validation of Sorrento’s worth will have to arrive from a partnering deal with a major pharma. We don’t see this on the immediate horizon; a lucrative deal usually requires clinical data.
Sorrento has a couple assets that are approaching mid-stage and late-stage clinical development including its CAR-T and Biosimilar candidates.
Equipment & Employees
During our visit, it was clear that Sorrento’s cash infusion has enabled the acquisition of capital assets and personnel.
1) The equipment is state of the art, equivalent to that owned by large biotechs and pharmas – This expensive lab equipment isn’t often see with other developmental stage biotechs. This should speed the development of candidate biologics.
2) The continued growth in lab personnel is noted as well as the educational and professional pedigree – We have written that Sorrento has experienced robust personnel expansion so it may execute on the development of its many programs. This has clearly continued. Most large labs are filled with “techs” to perform routine, repeated tasks. That’s appropriate. Sorrento was a bit different. Many of Sorrento's staff are scientists - tjeu hail from some of the best labs in the country. They should help push the development of Sorrento’s advanced technologies.
3) There is ample space for continued expansion – In Sorrento’s latest 10-Q, the plan is to continue hiring for the next year.
4) There is the potential for vertical integration – While CEO Ji did not commit to any future plans, the current construction should support enough commercial capacity for cell therapy products for the foreseeable future.
Major Ownership of SRNE
As of December 17, 2015, Patrick Soon-Shiong personally owned 873,938 shares of SRNE, but also controls SRNE shares through a fund (Cambridge Equities LP), limited liability company (MP 13 Ventures LLC), and a foundation (Chan Soon-Shiong Family Foundation). He thus controls 22.3% of SRNE. He also has majority control of NantKwest, a major partner for Sorrento, as well at the “Nant family of companies”.
Soon-Shiong is the inventor of Abaxane and founder of Abraxis BioScience. Along with Soon-Shiong’s other investments, Abraxis’ acquisition gave him the title “Richest Man in Los Angeles”. His stated intent is to drive medicine forward through his Nant companies. As with any large, fast-growing endeavor, we expect some initial chaos at the Nant companies. A few of his professional acquaintances have described him as a gifted biotech inventor – they don’t expect him to be a one-shot wonder.
Sorrento has “mutual interests” with Soon-Shiong through a number of his companies.
Patrick Soon-Shiong (PSS) & Sorrento Therapeutics
Company
Location
Relationship
Sorrento Therapeutics
(SRNE)
San Diego, CA
Through various entities, PSS owns 22.3% of SRNE.
NantKwest (NK)
Carlsbad, CA
SRNE owns 19.9% of NK, while PSS is its CEO and majority owner. SRNE has partnered with NK towards the development of its proprietary CAR-directed, Natural Killer cells called CAR.TNK.
TNK Therapeutics
San Diego, CA
Subsidiary of SRNE to develop CAR.TNKs and CAR-Ts, using Sorrento’s G-MAB library to create new CAR constructs.
In August 2015, SRNE and TNK exclusively licensed the NanoVelcro Circulating Tumor Cell profiling assay (the “Technology”) from Cytolumina Technologies Corp. (“CTC”) and Fetolumina Technologies Corp. (“FTC”). TNK shall acquire 4.166% of the capital stock of each of CTC and FTC for an aggregate purchase price of $5M.
TNK, on the one hand, and CTC and FTC, on the other hand, shall share the profits from the net sales of the Technology for any cell based therapies in the TNK Field or SRNE antibody on a 50/50 basis.
NantCell,
NANTibody
Culver City, CA
San Diego, CA
In April 2015, SRNE granted an exclusive license to NantCell covering patents, know-how, and materials related to certain antibodies, anti-body drug conjugates (ADC) and two CAR-TNK products.
NantCell agreed to pay a royalty not to exceed 5% to SRNE on any net sales of products. In addition, SRNE received an upfront payment of $10M and 10M shares of NantCell common stock to SRNE (valued at $100M based on a recent equity sale of NantCell common stock to a third party).
SRNE’s ownership interest in NantCell does not provide SRNE with control or the ability to exercise significant influence.
In April 2015, SRNE and NantCell also established a joint venture NANTibody, as a stand-alone biotech with $100M initial joint funding. NantCell owns 60% and contributed $60M to NANTibody. SRNE owns 40% of NANTibody and contributed $40M. NANTibody will develop multiple cancer mAbs, including but not limited to anti-PD-1, anti-PD-L1, anti-CTLA4 mAbs, and other checkpoint mAbs as well as antibody drug conjugates (ADCs) and bispecifics.
NantBioScience, NantStem
Culver City, CA
San Diego, CA
In July 2015, SRNE and NantBioScience established a new joint venture called NantCancerStemCell LLC, or NantStem, as a stand-alone biotechnology company with $100M in initial joint funding. NantBioScience contributed $60M cash to NantStem for a 60% ownership in NantStem and SRNE was obligated to make a $20M cash contribution to NantStem for 20%.
SRNE also issued a call option on shares of NantKwest (NK) that it owned to Cambridge Equities, LP (a PSS fund). The call option to Cambridge is on up to 2.0M shares of NK held by SRNE (the Option Agreement). The Option Agreement gives Cambridge the right to purchase up to 2.0M shares at a price of $15.295 from time to time in Q1-2016.
NantStem will develop small molecules against targets which may address important drivers of cancer growth including cancer stem cells. Sorrento will contribute specified small molecule programs (lead inhibitors of the proto-oncogenes c-Myc, and the master metabolism regulator HIF-1 alpha, and an inducer of the tumor suppressor cytokine TRAIL).
NantPharma
Lincolnshire, IL.
In July 2015, NantPharma purchased the Cynviloq program from SRNE. SRNE may receive up to $620M in regulatory milestone payments and up to $600M in sales milestones, of which $45M has already been disbursed. SRNE will also receive “transfer payments” (“think single digit royalties”) on manufactured units of Cynviloq. During the first three years after closing, SRNE has the option to co-develop and/or co-market Cynviloq on terms to be negotiated
Other major holders include:
Opko Health, Inc 5.7%
venBio Select Advisor 5.7%
Cormorant Asset Mgt 5.6%
Wildcat Capital Mgt 4.5%
Next Part
Part 2 of BioWatch’s series on Sorrento (SRNE) will summarize its BioBetter program.
(At the time this post was written, one or more BioWatch writers held a long position in SRNE)
This blog post is from The Biotech Watcher: http://alanhobbes.blogspot.com/?view=magazine
And about us, see http://alanhobbes.blogspot.com/2014/12/welcome-to-my-personal-thoughts-about.html
Posted Yesterday by Alan Leong
Labels: $SRNE BioBetters mAb ownership Soon-Shiong Sorrento Therapeutics Update
0 A
Henry Ji Speaking today at the large biotech conference in San Fran today, he has 3 presentations this week. He has the lead in 9am slot today. In anticipation of that, SRNE released phase 3 trial study results on some of the biosimilars they purchased from Mabtech. Impressive results making them biobetter drugs. In addition, a 2nd news release on a JV with the Swiss research company that discovered the NK cells doing research on how best to use them. Should be an interesting day/week for SRNE.
I think it has more to do with the filings yesterday on Edgar by both SRNE and PSS. These are filings seem to indicate some type of takeover or merger according to at least one analyst.
A interesting new Seeking Alpha article out today about NK and PSS's option deal with SRNE for NK stock.
http://seekingalpha.com/article/3633036-nantkwest-filing-may-be-offering-a-glimpse-into-the-future?app=1&auth_param=kd59j:1b3f3ve:34985637fc701066d06a86eb25fbfee6&uprof=45
I think quarterlies will be out tomorrow or soon. They have made so many deals over the summer, kind of hard to envision cash position. Hopefully, they will show plenty of cash.
There was an article this summer in Seeking Alpha on SRNE before some of the latest deals and I think the author pegged it. He said, do not look at SRNE for its science but for its deal making and this was mainly for their deals with PSS. They seem to be acting more as an investment banker/venture capitalist/gold-silver streaming company all in one. I think they are going to sell off or JV the rights to the biosimilar/betters and retain royalty interests and the same could be said about many of the products in their pipeline. The new COO supposedly has lots of China ties and was surprised that SRNE was able to negotiate the deal for the 4 biosimilars and one of the reasons he went with them. At least one analyst thinks SRNE could sell the rights to the Remicade biobetter for $billions as it supposedly is the only one already in existence.
If the cell penetrating technology from City of Hope were to be proven to work, it would be worth many billions as it is the holy grail of biotech and every company would want the rights to it.
It is hard to see them missing on all of their bought deals, and they only need to hit on one of their potential blockbusters. jmho
If anyone still following SRNE they updated their Corporate Presentation. They have a lot going on and they show the targets for their biosimilars and much more. Let's see if it catches the attention of the market.
Great News for the new week. Maybe this will stop the slide? What do you think? I think Dr. Ji is a great deal maker.
.................................................................
Sorrento's TNK Therapeutics Subsidiary Acquires Multiple Clinical Stage CAR-T Immunotherapy Programs
Ticker Symbol: U:SRNE
Sorrento's TNK Therapeutics Subsidiary Acquires Multiple Clinical Stage CAR-T Immunotherapy Programs
PR Newswire
SAN DIEGO, Aug. 10, 2015
SAN DIEGO, Aug. 10, 2015 /PRNewswire/ -- Sorrento Therapeutics, Inc. (NASDAQ: SRNE) (Sorrento) announced today that its wholly-owned subsidiary, TNK Therapeutics, Inc., has acquired multiple preclinical and clinical stage chimeric antigen receptor (CAR)-T immunotherapy programs as well as underlying CAR-T technology through the acquisition of two privately-held biotechnology companies. The CAR-T programs focus on targeting solid tumors as well as infectious diseases.
Sorrento Therapeutics, Inc.
"We are very pleased to enter the dynamic CAR-T immunotherapy field with these clinical stage assets targeting solid tumors, an area of great unmet medical need," said Dr. Henry Ji, President and CEO of Sorrento. "Especially exciting is the potential of combining the CAR-T therapies with Sorrento's immune-oncology programs, such as anti-PD1 and anti-CTLA4 monoclonal antibodies (mAbs). We recently in-licensed late clinical stage biobetter mAbs of the marketed antibodies infliximab, cetuximab, and basiliximab, as well as a biosimilar mAb of omalizumab. Utilizing these assets, combination therapies of our biobetter mAb of basiliximab, an anti-CD25 mAb that has been used to target and deplete immunosuppressive regulatory T cells1, or cetuximab, an anti-EGFR (epithelial growth factor receptor) mAb, may work synergistically with our CAR-T and CAR.TNK programs for the treatment of solid tumors. With these acquisitions of clinical and pre-clinical CAR constructs, TNK Therapeutics is now positioned to accelerate the development of in-house adoptive immunotherapies, including the "off-the-shelf" CAR.TNK programs in our exclusive partnership with NantKwest. This breadth of complementary clinical programs and enabling technologies truly positions TNK Therapeutics to be a leader in the field of adoptive immunotherapies."
Information about these CAR-T programs, underlying technologies as well as the biosimilar/biobetter antibodies will be detailed in Sorrento's updated corporate presentation later this month.
About Sorrento Therapeutics, Inc.
Sorrento is a clinical stage biopharmaceutical company developing new treatments for cancer and associated pain and inflammation and autoimmune diseases. Sorrento recently licensed multiple late-stage biosimilar and biobetter antibodies for oncology and inflammation diseases for the US, European and Japanese markets. Sorrento recently sold the rights to Cynviloq, which successfully completed the TRIBECA study, to NantPharma. The company is also developing resiniferatoxin (RTX), a non-opiate TRPV1 agonist to treat terminal cancer patients suffering from intractable pain.
In December 2014, Sorrento and NantWorks formed a global joint venture, now called Immunotherapy NANTiBody, LLC, to focus on immunotherapies for cancer. Also in December 2014, Sorrento and Conkwest, Inc., now renamed as NantKwest, Inc., an immuno-oncology company developing proprietary Neukoplast®, a Natural Killer (NK) cell-line based therapy, entered into an agreement to jointly develop CAR.TNK (Chimeric Antigen Receptor Tumor-attacking Neukoplast) immunotherapies for the treatment of cancer and infectious diseases. In March 2015, Sorrento entered into a global collaboration with NantCell, a NantWorks company, to discover and develop immunotherapies against tumor neo-epitopes. In July 2015, Sorrento and NantBioScience, Inc., a subsidiary of NantWorks, established a joint venture, called NantCancerStemCell, LLC to focus on the development of "first-in-class" small molecules against targets which may address important drivers of cancer growth including cancer stem cells.
Forward-Looking Statements
This press release contains forward-looking statements related to Sorrento Therapeutics, Inc. under the safe harbor provisions of Section 21E of the Private Securities Litigation Reform Act of 1995 and subject to risks and uncertainties that could cause actual results to differ materially from those projected. Forward-looking statements include statements about Sorrento's prospects, including, but not limited to any statements about the chimeric antigen receptor (CAR) T cell programs; potential combination therapies, Sorrento's expectations for adoptive cellular immunotherapies, Sorrento's collaborations with NantKwest, NantCell, NantPharma and NantBioScience, and the development of adoptive immunotherapies and the biosimilar/biobetter programs; Sorrento's ability to leverage the expertise of its employees and partners to assist the company in the execution of its strategies; Sorrento's advances made in developing RTX, CAR.TNKs and human monoclonal antibodies using its proprietary G-MAB fully human antibody technology, if any; and other matters that are described in Sorrento's Annual Report on Form 10-K for the year ended December 31, 2014, and subsequent Quarterly Reports on Form 10-Q filed with the Securities and Exchange Commission, including the risk factors set forth in those filings. Investors are cautioned not to place undue reliance on these forward-looking statements, which speak only as of the date of this release and we undertake no obligation to update any forward-looking statement in this press release except as required by law.
Sorrento, G-MAB, CAR.TNK, TNK Therapeutics, and the Sorrento logo are trademarks owned by Sorrento Therapeutics, Inc.
All other trademarks and trade names are the property of their respective owners.
1 Int J Oncol. 2009 Feb;34(2):563-72
Logo - http://photos.prnewswire.com/prnh/20150105/167173LOGO
NEWS TODAY, NEW DEAL WITHOUT PSS UNLESS IT WAS HIS CONNECTIONS;
Sorrento In-Licenses Four Late-Stage Clinical Biobetter and Biosimilar Antibodies
2015-08-03 06:00 ET - News Release
SAN DIEGO, Aug. 3, 2015 /PRNewswire/ -- Sorrento Therapeutics, Inc. (NASDAQ: SRNE; Sorrento) announced today that it has entered into an exclusive licensing agreement to develop and commercialize multiple prespecified and undisclosed biosimilar or biobetter antibodies from Mabtech Limited, a holding company for premier antibody development and manufacturing companies in China.
Under the terms of the agreement, Sorrento will develop and market these 4 monoclonal antibodies (mAbs) for the North American, European and Japanese market. Each of the mAbs has completed a Phase III study; two are currently in registration for marketing approval in China, while the other two are under data analyses for subsequent NDA submission in China. One of the biobetter mAbs is based on a Top 10-selling oncology drug and the other a Top 5-selling antibody to treat auto-immune diseases. Together these 4 mAbs target an established market with combined annual global sales in 2014 in excess of $13 Billion. Based on a study by GBI Research[1] the global biosimilar market could reach an estimated $55 Billion by 2020.
"Combining our expertise in immunotherapy and drug development with Mabtech's experience in antibody development and commercial cGMP manufacturing positions the two companies well to increase access worldwide to biosimilars and improve global human health," said Dr. Henry Ji, President and Chief Executive Officer at Sorrento. "We look at this collaboration as a transformational event for Sorrento and are excited about its potential to complement our expanding and deep internal biologics portfolio. With this exclusive license agreement, Sorrento takes a significant step toward becoming a major player in the biopharmaceutical industry."
"We welcome the opportunity to work with Sorrento Therapeutics to bring our antibody products to a global market beyond the Greater China territory. During our discussions with the Sorrento team it became apparent that Sorrento is committed to be a leader in the antibody immunotherapy space," added Mr. Zhen Jiao, Chairman of Mabtech and Chief Executive Officer of CDH Investments, the top private equity fund in Asia with more than $10 billion under management. "We believe that, together with Sorrento's strong commitment and our vast resources, we will be able to provide much needed immunotherapies to patients and healthcare providers worldwide in an expedited and efficient manner."
About Sorrento Therapeutics, Inc.
Sorrento is a clinical stage oncology company developing new treatments for cancer and associated pain. Sorrento recently sold the rights to Cynviloq™, which successfully completed the TRIBECA™ study, to NantPharma. The company is also developing resiniferatoxin (RTX), a non-opiate TRPV1 agonist currently in a Phase 1/2 study at the NIH to treat terminal cancer patients suffering from intractable pain.
In December 2014, Sorrento and NantWorks formed a global joint venture, now called Immunotherapy NANTiBody, LLC, to focus on immunotherapies for cancer. Also in December 2014, Sorrento and Conkwest, Inc., now renamed as NantKwest, Inc., an immuno-oncology company developing proprietary Neukoplast®, a Natural Killer (NK) cell-line based therapy, entered into an agreement to jointly develop CAR.TNK™ (Chimeric Antigen Receptor Tumor-attacking Neukoplast) immunotherapies for the treatment of cancer and infectious diseases. In March 2015, Sorrento entered into a global collaboration with NantCell, a NantWorks company, to discover and develop immunotherapies against tumor neo-epitopes. In July 2015, Sorrento and NantBioScience, Inc., a subsidiary of NantWorks, established a joint venture, called NantCancerStemCell, LLC to focus on the development of "first-in-class" small molecules against targets which may address important drivers of cancer growth including cancer stem cells
I think a lot of SRNE $ went into NK. The 30 mins after NK opened today SRNE lost more than $3.00....... at the same time they gained at least $200 mil in book value from the NK stock.
NK was overpriced at $25 and oversold by 1.3mil shares and raised $207 mil. A biotech IPO record, according to one of the many releases. Should mean good news for SRNE tomorrow as the NK shares are on the books for $21.5 mil cost basis and now will be on the books for market basis which should be close to $400mil in assets added tomorrow. I think the strong demand will mean NK will close maybe $60+
Here is good write up prior to knowing the price but released earlier today, it meantions SRNE as a 8% shareholder but that will now be closer to 7% with the added shares and I think the underwriters still have 1.05 mil shares to sell at IPO price:
http://www.equities.com/editors-desk/investing-strategies/ipo/nantkwest-nk
Seem's this is the closest thing available for now. NK S-1 lists side deals with several of these subsidiaries.
http://www.nantworks.com/
I bought calls when around $15 the Aug were less than a $1.00 and Dec less than $2.00 both were up 500% or so. I bought both over several days and hoping SRNE moves big with the IPO as I want to unload the Aug calls. You know PSS mentioned taking Nantworks public by years end. There are now March calls, if SRNE moves big with this IPO it should move up with the next as isn't the JV deal with Nantworks?
NK getting big press. IBD article below. Anyone think the recent selling was due to raise cash to buy NK? I sold options to buy but have held my shares. I am thinking some may have taken SRNE profit to buy NK.
I think NK market cap will reach $5bil or maybe more quick, so 7.3% of that is getting close to $400mil in assets for SRNE. I do not know what they have it on the books for now, anyone know?
http://www.nasdaq.com/article/biotech-ipo-boom-gaining-thrust-with-nantkwest-debut-cm500848
Wow Heidi, Guess now you can paint your "hippy" barn. When were they .10? That is patience, and % wise one of the best returns I have seen.
I was lucky enough to listen to Chen Lin and loaded up on both Aug and Dec 20 calls when stock was around $15 before news of the JV with Nantworks, sold about 40% of them and have more than covered my cost. They are off big since late morning yesterday. I did sell a block then.
I will be watching closely next week to see what effect NK has on SRNE.
NK IPO is Tuesday, announced today and SRNE is down???? I am not sure that the SRNE will track with NK, it should go up but I am going to try and buy some NK early,
Another Good Article, what is consensus here? Would it make $ense to sell some SRNE at a nice profit and try to get into NK on the open? Anyone thinking of doing this? I am thinking NK will reach $50+ first day, since Seres did last month and I think their IPO price was $18 and if memory serves me, it opened at $28.50 just after 11 and I missed my $28.00 opening limit order and did not want to chase it.
http://www.nanalyze.com/2015/07/dr-soon-shiongs-car-t-ipo-nantkwest/
Patrick Soon-Shiong's recent ventures: http://www.bioportfolio.com/news/article/2401199/Sorrento-Therapeutics-SRNE-and-NantKwest-NK.html
Patrick Soon-Shiong's recent ventures: http://www.bioportfolio.com/news/article/2401199/Sorrento-Therapeutics-SRNE-and-NantKwest-NK.html
Here is the IPO presentation. I would think after seeing this that the IPO will go very well.
So General,
What is your thoughts after today. How long can this mo go? I am assuming 5 to 7 trading days to the NK IPO. I am thinking some of the analysts are going to have to take a look at this large spike and do some updates soon. There was one analyst that recently put a $33 target on this and the stock is now above all the targets but the $33.
In looking a little more. The largest trade of the day was just under $600k @ near the end of the day, it will be interesting to see what tomorrow brings. This should bring more attention tomorrow, be interesting to see if the Mo can continue. Every chart indicator seems to say buy but....
Trades for U:SRNE on 20150716 - 100 trades displayed
Time ET Ex Price Change Volume
15:50:18 Q 21.55 3.16 27,419
12:21:38 Q 18.90 0.51 22,159
15:47:05 Q 20.84 2.45 14,300
15:55:34 Q 21.36 2.97 11,800
10:43:48 Q 19.01 0.62 10,300
14:16:28 Q 20.00 1.61 10,000
15:48:06 Q 20.9136 2.5236 10,000
I would not be surprised to see another filing and the IPO oversubscribed as has happened a lot recently including on DNAI which opened today.
Think the NK IPO hype started this rally (19%) and the Mo watchers piled in? There was very heavy volume the last hour, where you would normally see a lot of 100's per min we see this, note the volume picking up near the close:
15:00 20.33 20.37 20.3032 20.37 15 3253
15:01 20.38 20.50 20.36 20.50 25 3550
15:02 20.501 20.60 20.501 20.60 19 3400
15:03 20.60 20.665 20.58 20.5907 12 1822
15:04 20.655 20.655 20.53 20.65 67 12190
15:05 20.64 20.662 20.618 20.65 7 1400
15:06 20.60 20.605 20.49 20.54 12 2996
15:07 20.58 20.63 20.56 20.61 8 2200
15:08 20.65 20.72 20.60 20.64 31 6208
15:09 20.6479 20.72 20.632 20.72 17 8300
15:10 20.67 20.75 20.67 20.70 32 5100
15:11 20.68 20.72 20.67 20.67 7 1300
15:12 20.67 20.75 20.67 20.75 26 5432
15:13 20.75 20.89 20.74 20.89 35 9958
15:14 20.89 20.90 20.77 20.785 26 19890
15:15 20.75 20.82 20.75 20.77 11 2600
15:16 20.77 20.90 20.77 20.86 21 3900
15:17 20.90 20.95 20.85 20.89 21 6301
15:18 20.87 20.87 20.77 20.80 14 3350
15:19 20.80 20.80 20.77 20.80 10 3600
15:20 20.80 20.80 20.63 20.637 16 2830
15:21 20.695 20.695 20.6108 20.68 10 2500
15:22 20.645 20.76 20.618 20.76 26 4720
15:23 20.71 20.72 20.67 20.67 7 700
15:24 20.6699 20.70 20.658 20.68 10 2400
15:25 20.68 20.68 20.66 20.67 10 2400
15:26 20.66 20.74 20.64 20.71 27 4875
15:27 20.70 20.73 20.70 20.72 12 1200
15:28 20.722 20.76 20.72 20.74 13 1546
15:29 20.7265 20.74 20.71 20.71 4 3999
15:30 20.74 20.82 20.7196 20.817 58 10500
15:31 20.8199 20.90 20.80 20.86 62 12800
15:32 20.8678 20.90 20.8678 20.90 14 2325
15:33 20.86 20.90 20.766 20.766 15 2800
15:34 20.841 20.841 20.77 20.825 20 7850
15:35 20.79 20.8521 20.79 20.8521 6 1200
15:36 20.86 20.88 20.81 20.87 36 5052
15:37 20.87 21.00 20.85 20.93 48 16384
15:38 20.9606 21.00 20.93 20.97 31 11818
15:39 20.98 21.10 20.95 21.0785 32 15205
15:40 21.11 21.23 21.10 21.20 61 15721
15:41 21.15 21.20 21.10 21.10 70 9100
15:42 21.15 21.15 21.10 21.15 56 7600
15:43 21.11 21.15 21.01 21.044 72 22158
15:44 21.08 21.15 21.08 21.09 27 6950
15:45 21.15 21.20 21.12 21.16 50 12073
15:46 21.16 21.165 21.10 21.10 61 9648
15:47 21.11 21.1419 20.84 21.1419 131 37068
15:48 21.1101 21.19 20.9136 21.15 42 15400
15:49 21.23 21.30 21.12 21.19 103 20624
15:50 21.19 21.56 21.13 21.38 270 76202
15:51 21.38 21.45 21.32 21.45 63 10340
15:52 21.44 21.56 21.31 21.46 90 17132
15:53 21.45 21.47 21.38 21.44 112 16097
15:54 21.45 21.47 21.38 21.47 97 12148
15:55 21.5175 21.5175 21.26 21.40 156 34773
15:56 21.41 21.51 21.40 21.48 58 13515
15:57 21.48 21.78 21.48 21.645 175 34209
15:58 21.64 21.65 21.53 21.65 124 34436
15:59 21.66 22.02 21.66 21.88 149 53690
They priced the NK IPO today $20-$23 and to close (open) week of the 27th. If NK reaches $50, it would be worth about $280mm to SRNE in value of their NK stock. Another S-1 filed for NK today showing all and the revised % that SRNE will own. A little over 7% now.
NantKwest (previously Conkwest) in their amended S-1 doubled the number of shares being offered in their IPO from 100mil to 200mil. Think that is an indication of strong demand to a company controlled by Patrick Soon-Shiong?
Celgene backing Soon-Shiong's cancer biotech IPO, now dubbed NantKwest
July 14, 2015 | By John Carroll
Now that billionaire entrepreneur Patrick Soon-Shiong has taken control of tiny ConkWest and steered it toward a $173 million IPO, he's adding a makeover to the name that will fit more easily under the fast-growing NantWorks umbrella he's created. In an amended IPO filing ConkWest is now named NantKwest, which will look to trade under the "NK" symbol on Nasdaq. And Soon-Shiong is bringing some close associates in on the deal, with Celgene (which acquired Abraxis from Soon-Shiong for $3 billion) agreeing to buy $17 million worth of shares on the debut and funds at Franklin Templeton committing to a $45 million buy. For the record, the little immuno-oncology player had spent about $46 million by the time Soon-Shiong stepped in earlier this year, raised cash for the company and pointed it to the public market in record time. Filing
They Doubled the # of NK shares to be issued
.
There is really a lot of info in the prospectus. I note that they amended the # of common shares to be issued from 100mil to 200mil the # of preferred shares stayed @20mil. Think that means the demand is much greater than originally planned or why double the # of shares? also, how does that and the stock split affect SRNE's % of ownership? SRNE has 85%+ more shares now, I guess to make up for this?
ORIGINAL ARTICLE IV
Classes of Stock. The total number of shares of capital stock that the Company shall have authority to issue is 120,000,000, consisting of 100,000,000 shares of Common Stock, $0.0001 par value per share (“Common Stock”), and 20,000,000 shares of Preferred Stock, $0.0001 par value per share (“Preferred Stock”).
AND THE AMENDED
5. Article IV of the Certificate is hereby amended and restated in its entirety to read as follows:
“Upon the effectiveness of this Certificate of Amendment, each outstanding share of Common Stock or Preferred Stock of the Corporation shall be split into 1.8515 shares of Common Stock (the “Forward Split”). The Forward Split shall occur automatically without any further action by the holders of the shares affected thereby and whether or not the certificates representing such shares are surrendered to the Company.
After giving effect to the Forward Split, the total number of shares of stock that the Corporation shall have authority to issue is Two Hundred Twenty Million (220,000,000), consisting of Two Hundred Million (200,000,000) shares of Common Stock, $0.0001 par value per share, and Twenty Million (20,000,000) shares of Preferred Stock, $0.0001 par value per share
Here are the 3 largest Biotech IPO's so far this year. I think NK has the possibility of cracking this top 3 list, does anyone know if Dr. S-S's recent crosstrade counts towards the IPO process and would be in addition to money raised in the IPO which I think will be substantially oversubscribed?
#3. Galapagos
Amount: $316.7 million in gross proceeds (approximately €278.7 million)21
Date: May 19
Shares/ Price per share: 7,532,499 shares at $42.0522
Symbol/Market: GLPG on NASDAQ Global Select Market, as well as on Euronext Amsterdam and Euronext Brussels
#2. Axovant Sciences
Amount: $362 million in gross proceeds prior to deducting underwriting discounts and commissions and estimated offering expenses payable by company23
Date: June 16
Shares/Price per share: 24.15 million shares at $1524
Symbol/Market: AXON on New York Stock Exchange
#1. 3SBio
Amount: $534 million in net proceeds after deducting underwriting fees and estimated expenses in connection with the offering
Date: June 24
Shares/Price per share: 853,052,440 shares at HK$9.10 ($1.17)25
Symbol/Market: 1530 on Hong Kong Exchange
I assume the NK news is what caused the SRNE price increase to a new 52 week high before pulling back. It looks like there is a lot in interest in IPO and I think it was a smart move for the name change. There is a lot of info in the amended prospectus including this stock split shown below. How will this affect SRNE interest in the stock if at all and would this be because of them knowing the initial price and some formula that must be used? Will SRNE still own about 8% of shares after the opening? I think NK could be trading as soon as next week.
"On July 10, 2015, we effected a 1.8515-for-1 forward stock split of our outstanding common stock. This prospectus gives retroactive effect to the split for all periods presented."
This is big news and I am surprised stock not up more. I saw an estimate that the Dr is now at $15bil net worth and think that will change quickly with this IPO and his Nantworks IPO which he hopes to have out by years end.
I am wondering when the market is going to catch on how close SRNE is related to all of the Dr's companies. SRNE even gets royalties from the NK products even if SRNE is not part of the product. I think the Conkwest IPO could be before months end. Some of the recent biotech's were within 30 days of the S1 filings.
Conkwest filed an amended S1 on July 2nd, the requested symbol is NK and SRNE is mentioned often in the very long prospectus. If you go to SEC Edgar and search under conkwest you can read the very detailed prospectus but have to be a scientist to understand much of it. I think that NK could be trading in as little as two weeks from now.
News
SRNE added to the Russell Index's, the 2000, 3000 and Global. Was already in their microcap index.
If you read the S-1 for Conkwest, you will see many references to SRNE (well in excess of 100) and including lots of profit sources/royalties for SRNE from Conkwest. I do not think the market has caught on yet to how closely they are entwined.
http://www.sec.gov/Archives/edgar/data/1326110/000119312515229037/d917371ds1.htm
I am thinking the IPO will open on NASDAQ by Aug 1 and will be one of the hottest IPO's of the year. Do others agree with this? I would like to hear the thoughts of others on this.
There seems to be some profit taking the past couple of days and maybe a good time to pick up some more shares.
Anyone following today's news RE: Conkwest IPO and SRNE's close relationship? With all of the media coverage of Conkwest such as 60 minutes, I think IPO will be over subscribed and a big opening hit greatly benefiting SRNE.
Any thoughts by anyone on 6 month potential of SRNE?
FINALLY HARD FACT GOOD NEWS!
Stellar Biotech to provide KLH for BiovaxID treatment
Ticker Symbol: C:KLH
Stellar Biotech to provide KLH for BiovaxID treatment
Stellar Biotechnologies Inc (C:KLH)
Shares Issued 79,421,650
Last Close 10/22/2014 $1.39
Thursday October 23 2014 - News Release
Mr. Mark McPartland reports
STELLAR BIOTECHNOLOGIES AND BIOVEST INTERNATIONAL SIGN KLH SUPPLY AGREEMENT FOR BIOVAXID IMMUNOTHERAPY FOR FOLLICULAR NON-HODGKIN'S LYMPHOMA
Stellar Biotechnologies Inc. and Biovest International Inc. have executed a definitive supply agreement to meet Biovest's requirements for keyhole limpet hemocyanin (KLH) for use in Biovest's BiovaxID active immunotherapy to treat follicular non-Hodgkin's lymphoma.
Stellar is a leader in sustainable manufacture of KLH, an immune-stimulating protein widely used as a carrier molecule in active immunotherapy drugs in development for certain cancers and other diseases. Stellar manufactures its KLH products under the brand Stellar KLH.
Biovest is a biotechnology company developing and commercializing BiovaxIDtrademark (dasiprotimut-T), an active immunotherapy to treat follicular non-Hodgkin's lymphoma. BiovaxIDtrademark combines autologous heterohybridoma-derived tumor idiotype protein coupled to KLH as the carrier molecule. BiovaxIDtrademark has successfully completed Phase 2 and Phase 3 clinical trial development and is currently the subject of a Marketing Authorization Application (MAA) under review by the European Medicines Agency (EMA).
The purpose of the supply agreement is to establish the terms for the production and supply of Stellar KLH? to Biovest, for use as an active component in BiovaxIDtrademark immunotherapy vaccine in both commercial distribution as well as for future clinical trials.
The supply agreement requires Stellar to deliver Stellar KLH? to Biovest compliant with cGMP standards required for Biovest's ongoing development and as an anticipated commercial supply. Biovest is obligated to purchase Stellar KLH? at agreed forecasted quantities and prices. The supply agreement has an initial three-year term, which may be renewed by Biovest for additional one-year periods.
The supply agreement provides for Stellar and Biovest to consummate a separate quality agreement, within three months, to list the quality aspects and procedures relating to manufacture and release of the cGMP-compliant Stellar KLH?. Biovest will appoint Stellar as exclusive supplier of KLH in connection with the potential future commercialization of BiovaxIDtrademark, subject to negotiation and execution of commercial production and supply terms.
"Stellar's key growth initiative is to leverage our Stellar KLH? technology into multiple clinical pathways and the BiovaxIDtrademark program is a good example of the value of our core business for this purpose," said Frank Oakes, President and CEO of Stellar Biotechnologies, Inc. "There are many new KLH-based immunotherapies advancing in clinical trials and we are positioning Stellar to be the leading company capable of delivering the scalable, sustainable supplies of KLH that will be needed by these pharmaceutical pipelines."
"We are pleased to collaborate with Stellar Biotechnologies to continue the clinical progress of our active immunotherapy (BiovaxID) and are proud to work with Stellar in anticipation of our future commercial success," said Carlos Santos, Chief Executive Officer of Biovest. "BiovaxID immunotherapy has demonstrated ability in long-running clinical trials to elicit potent anti-tumor immune responses and extend remission duration in patients suffering from follicular non-Hodgkin's lymphoma. If approved, we anticipate that this product will offer an innovative adjuvant/consolidation vaccine strategy for patients with this disease."
About Biovest International, Inc.
Biovest International, Inc. is a pharmaceutical company focused in the field of active personalized immunotherapy development targeting life-threatening cancers of the blood system, marketing state-of-the-art bioreactors, and providing a full range of custom biomanufacturing services. Biovest's lead personalized cancer vaccine candidate BiovaxIDtrademark is an autologous active immunotherapy (personalized cancer vaccine) that targets follicular non-Hodgkin's lymphoma, mantle cell lymphoma, and potentially other B-cell malignancies. BiovaxIDtrademark has undergone three clinical trials conducted in collaboration with the U.S. National Cancer Institute (NCI) that have demonstrated BiovaxIDtrademark's ability to increase the duration of cancer remission following chemotherapy and to induce immune responses which correlate highly with long-term survival. Biovest is currently in the process of pursuing European marketing approval for BiovaxIDtrademark.
I am a little late in replying, but I think that IF KLH is used in any vaccine and needed in quantity it is pretty obvious that restrictions will be put out by the US or CA governments to protect the mollusk. The science article I just posted referred to this.
The SA article says the other biotech company can extract KLH without killing the animal. I doubt this is true or if whoever harvests the KLH even cares whether they kill the mollusk or not. If something is put on the endangered species list, it is totally hands off. You cannot touch it or affect it in any way whatsoever. A prime example is the Manatee, some tourist in FL posted video of herself petting and even attempting to ride a very tame wild manatee. It was obvious that she had no ill intent and did not harm the animal but the video became very public and she was arrested facing some serious charges. I did not hear the outcome and hopefully she did not do jail time, but you can bet if she was trying to extract the blood of the manatee, she would be in Federal prison.
Another Newsletter touting this company as a teaser to buy their newsletter. I received this in an e mail today. It is a very informative article and quite the pump, I assume their subscribers already may have been given the article recently and may have been partly responsible for the nice uptick yesterday. This will probably not post in a workable format, so here is the link....
http://www.angelnexus.com/o/web/65254
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Dear Reader,
Have you seen the rare footage I was able to capture?
If you remember where you were and how you felt after events like the moon landing and the killing of Osama bin Laden...
This video will leave you with similar effects.
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You should see this write up and video. I need to read this board more often. This is dated 10/16 by a Science reporter and very informative....
http://science.kqed.org/quest/video/in-rare-sea-snail-scientists-find-compound-that-could-help-cancer-patients/
KLH in Ebola Vaccine Research
This is interesting, just scroll to the 2 highlighted parts. In one of the vaccines used here 100% of the mice injected with the ebola virus lived. I read this as the 2 successful vaccines used KLH in formula, how do others interpret this?
Here is link for this article:
http://omicsonline.org/2157-2526/2157-2526-S1-006.php
esearch Article Open Access
Protective Immunodominant Zaire Ebolavirus Glycoprotein Epitope in Mice
Xiangguo Qiu1, Lisa Fernando1, Steven M. Jones1,2,3,4 and Judie B. Alimonti1,2*
1Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St. Winnipeg, Manitoba, R3E 3R2, Canada.
2Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, R3E 3R2, Canada
3Department of Immunology, University of Manitoba, Winnipeg, Manitoba, R3E 3R2, Canada.
4Cognoveritas Consulting Inc. A-137 Westchester Dr., Winnipeg, Manitoba, R3P 2G6 Canada.
*Corresponding author: Dr. Judie Alimonti
Special Pathogens Program, National Microbiology Laboratory
Public Health Agency of Canada, 1015 Arlington St.,
Winnipeg, Manitoba, R3E 3R2
Tel: 204-789-5998 or 789-5097
Fax: 204- 784- 2140
E-mail: judie.alimonti@phac-aspc.gc.ca
Received August 26, 2011; Accepted October 18, 2011; Published October 20, 2011
Citation: Qiu X, Fernando L, Jones SM, Alimonti JB (2011) Protective Immunodominant Zaire EbolavirusGlycoprotein Epitope in Mice. J Bioterr Biodef S1:006. doi:10.4172/2157-2526.S1-006
Copyright: © 2011 Qiu X, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Zaire Ebolavirus (ZEBOV) causes a highly lethal severe haemorrhagic fever in humans, for which there is currently no approved vaccine available. In this study, cytokine ELISPOT assays were used to determine the ZEBOVGP-specific cytokine profile and immunodominant T cell epitopes in VSV?G/ZEBOVGP immunized Balb/C mice. Splenocytes added to pools of overlapping peptides spanning ZEBOVGP stimulated a Th1 cytokine profile with the majority of the ZEBOVGP-specific splenocytes secreting IFN-g. The splenocytes produced the strongest IFN-g response to the immunodominant peptide HNTPVYKLDISEATQ, located in the cytopathic mucin domain of GP. The immunodominant epitope was then tested for its ability to induce a protective immune response in mice challenged with a lethal dose of mouse adapted-ZEBOV. Mice immunized with the peptide plus Freund’s adjuvant survived the challenge, and had a strong ZEBOVGP-specific T and B cell immune response. Identifying the ZEBOVGP-specific cytokine profile and immunodominant epitope will aid in determining the correlates of immune protection; allow for the development of assays to assess the efficacy of the VSV?G/ZEBOVGP vaccine in immunized individuals; and provide valuable information for the development of a subunit vaccine
Keywords
Cytokines; Ebola; Epitope; Glycoprotein; IFN-g; Mice; Mouse adapted-Ebola; T cell; Vaccine; VSV
Abbreviations
eVLPs: Ebola Virus Like Particles; i.p.: Intraperitoneally; KLH: Keyhole Limpet Hemocyanin; MA-ZEBOV: Mouse Adapted ZEBOV; NHP: Nonhuman Primate; NP: Nucleoprotein; PA: Peptide without Freund’s adjuvant; PF: Peptide with Freund’s adjuvant; PK: Peptide-KLH conjugate; PKF: Peptide-KLH conjugate with Freund’s adjuvant; VSV: Vesicular Stomatitis Virus; ZEBOV: Zaire ebolavirus
Introduction
Zaire Ebolavirus is a filovirus that causes a severe haemorrhagic fever in humans with a fatality rate of 50-90%. There are currently no vaccines or effective therapies commercially available. However, due to a rapid and high fatality rate there is need for a vaccine. We have generated a vaccine (VSV?G/ZEBOVGP) that uses a live attenuated recombinant Vesicular Stomatitis Virus (VSV) with the VSV glycoprotein replaced by the glycoprotein (GP) of ZEBOV [1]. In nonhuman primates (NHPs) the VSV?G/ZEBOVGP vaccine provides 100% protection against a lethal high dose ZEBOV challenge, and induces long lasting humoural and cellular immunity [2,3]. Although antibody and T cell immune responses have been generated against the ZEBOV GP and nucleoprotein (NP), numerous studies have demonstrated that GP is sufficient in conferring protection [4,5]. As the VSV?G/ZEBOVGP vaccine is effective in NHPs it would be a useful model in which to study the immunological mechanisms of protection. Therefore determining the cytokine profile and immunodominant T cell epitope would be instrumental in the first steps of identifying putative protective mechanisms for ZEBOV, and aid in developing assays to establish the efficacy of the vaccine in eliciting a protective immune response in immunized individuals.
One of the most effective assays for examining the correlates of immune protection is the ELISPOT assay. The ELISPOT assay is a reliable, reproducible assay for the determination of T cell responses after vaccination, and has been used for many clinical trials to evaluate the immunological efficacy of a vaccine [6-8]. The ELISPOT assay, which can quantify low frequency immune responses on a per cell basis, provides information on the frequency (clonal size), and the cytokine effector signature of the Ag-specific T cell pool in vivo for a primed immune response [9]. In this study, peptides spanning the entire region of ZEBOVGP were added to a cytokine ELISPOT assay in order to determine the ZEBOVGP-specific cytokine profile, as well as the immunodominant ZEBOVGP epitope in VSV?G/ZEBOVGP immunized mice. The immunodominant peptide was then tested in a mouse model for its ability to provide protection to a lethal challenge of mouse adapted-Zaire Ebolavirus (MA-ZEBOV).
Materials and Methods
Animals and ethics
Female Balb/C mice aged 6-8 weeks were obtained from Charles River (Quebec, Canada). The animal studies were approved by the Canadian Science Centre for Human and Animal Health Care Committee following the guidelines of the Canadian Council on Animal Care. The mice were housed in either the Biosafety level laboratory 2 (BSL2) or BSL4 in environmentally enriched sterile housing with food and water ad libitum.
Vaccine and peptides
The recombinant VSV?G/ZEBOVGP vaccine expressing the glycoprotein (GP) of ZEBOV (strain Kikwit) were generated using VSV (Indiana serotype) as described previously [1,10]. The 167 peptide library (Mimotopes, Australia) were dissolved in 80% Dimethyl sulfoxide (DMSO) and consisted of 15mers with 11 amino acid overlaps, and spanned the entire 676 amino acids of the ZEBOV, strain Kikwit 1995 GP. Initially, three peptide pools were made: pool 1 (peptides #1-56), pool 2 (peptides #57-112), and pool 3 (peptides #113- 167). Later a second batch of peptide pools were generated from pool # 2 (Table 1). The matrix was designed so that each peptide was present in two pools. In order for a response to an individual peptide to be considered positive it had to be positive in both pools. This allowed the identification of individual immunodominant peptides that could then be tested.
Table 1: Peptide Pool Matrixa.
Immunization and splenocyte preparation
The mice were acclimatized for 1 week prior to immunization. Mice received 2x104 pfu VSV?GZEBOVGP intraperitoneally (i.p.) at least one month prior to boosting with a second immunization. At 14 days after the boost, the spleen was homogenized by passing it through a wire mesh in RPMI 1640, strained through a 40 mm filter, then centrifuged at 450xg for 10 minutes, before resupension in RPMI 1640, 10% heat inactivated fetal bovine serum, 100 U penicillin, 100 mg streptomycin, 2 mM L-glutamine, 25 mM Hepes, non-essential amino acids, 1 mM sodium pyruvate, 50 mM ß-mercaptoethanol. They were strained again before addition to the ELISPOT assay plates.
ELISPOT assay
Mouse IFN-g, TNF, IL-2, IL-4, and IL-10 BD ELISPOT assays (BD Bioscience) were performed as per manufacturer’s instructions. Briefly, 5x105 splenocytes/well were incubated with 2 mg/ml of each ZEBOV GP peptide. For the positive controls the mitogens 0.5 mg/ml PMA plus 5 ng/ml Ionomycin was used for IFN-g, and IL-2; 1 mg/ml LPS was used for TNF and IL-10; and 5 mg/ml Con A was used for IL-4. For the negative control 80% DMSO was added at an equivalent volume as the peptides added. Spots were acquired by an automated AID ELISPOT Reader System (CTL, Cleveland, OH) using ELISPOT software version 3.5. The ELISPOT response was considered positive when the number of specific spots/million splenocytes was greater than 4 standard deviations above the average of the DMSO control wells. In addition, the spots in the DMSO negative control well had to be below 50. The results shown in the figures are after the DMSO background was subtracted for each mouse.
Murine protection study
Groups of Balb/C mice (n=7) were immunized subcutaneously with media only (medium), 50 mg of peptide (PF) with or without (PA) Freund’s complete adjuvant, or 50 mg of peptide conjugated to Keyhole Limpet Hemocyanin (KLH) with (PKF) or without (PK) Freund’s complete adjuvant. The mice were immunized twice more, 28 days apart, but with Freund’s incomplete adjuvant used in place of Freund’s complete adjuvant. A control group of mice were immunized intraperitoneally with 2 x 104pfu of VSV?G/ZEBOVGP (rVSV) 28 days before the challenge. At 28 days after the last immunization all groups of mice (n=5) were challenged intraperitoneally with 1000 LD50 of mouse adapted Zaire Ebolavirus (MA-ZEBOV) [11]. The survival of the challenged mice was followed for 28 days, which is 3 times longer than the length of time to death of the media only group. The splenocytes for the remaining 2 mice from each group were used in an IFN-g and IL-2 ELISPOT assay two weeks after the third immunization. The 2 mice in the positive control group (rVSV) which received only one immunization were re-immunized 28 days later and the splenocytes assayed two weeks later.
Indirect ELISA
The generation of the Ebola virus like particles (eVLPs) containing the ZEBOV GP1,2 strain Mayinga and VP40 was described previously [12,13], and the eVLPS were used as the antigen in the ELISA. There is only 11 amino acids difference between the Mayinga and Kikwit strains, and they are highly cross-reactive in this assay. High binding polysterene microtitre plates (Thermo) were coated with 60 ml of the eVLP diluted at 5 mg/ml in PBS for 1 hour at 37ºC. The wells were washed 4X with PBS, 1% Tween-20 then blocked with PBS, 2% skim milk (blocking solution) overnight at 4ºC. MAbs diluted in blocking solution (150ml) were added and incubated at 37ºC for 1 hour. After washing the plates 4X, the peroxidase-conjugated affinity purified goat-anti-mouse IgG detection antibody (Rockland) diluted 1:2000 in blocking solution was added for 1 hour at 37ºC. After washing 4X, the substrate (ABTS + H2O2, 100 ml/well) was added and the plates incubated at 37ºC. After 30 minutes, the reaction was read at room temperature with a spectrophotometer (Versamax microplate reader) at 405 nm. All samples were tested in triplicate.
Statistics
All statistics were performed using the Graph Pad Prism v4 software. T tests and Kaplan-Meier survival curve statistics compared the test mice to the media control mice.
Results
Determination of the cytokine profile and immunodominant ZEBOVGP epitope in splenocytes from VSV?G/ZEBOVGP immunized mice.
As the ELISPOT assay is a reliable assay for the determination of T cell immune responses to antigens in vaccines, the assay was used to determine what the cytokine profile and immunodominant ZEBOVGP epitope was in mice immunized with the VSV?G/ZEBOVGP vaccine. Initially, VSV?G/ZEBOVGP immunized splenocytes from Balb/C mice were added to a range of cytokine ELISPOT assays in order to determine which cytokines were predominantly invoked by the vaccine (Figure 1). For this initial assay 3 pools of 15mer peptides with 11 amino acid overlaps were generated spanning the entire ZEBOVGP. Each pool contained 56 peptides. The VSV?G/ZEBOVGP immunized splenocytes produced strong signals in pool #2 for the IFN-g, IL2, and TNF ELISPOT assays (Figure 1). However, there was a negative test result for the IL-4 and IL-10 assays as the signal was below the cutoff point for the wells containing the ZEBOVGP peptides. The positive controls for IL-4 and IL-10 worked confirming that the assay worked but the test results were negative. As the IFN-g ELISPOT assay produced the strongest and most reproducible response, this assay was then used to determine the immunodominant epitope.
Figure 1: ZEBOV GP-specific Cytokine ELISPOT Response. ZEBOVGP-specific cytokine secreting splenocytes were detected in 5 different cytokine ELISPOT assays at 14 days after boosting the VSV?G/ZEBOVGP immunized Balb/C mice. The splenocytes were stimulated with either peptide pool 1 (peptides 1-56), 2 (peptides 57-112), or 3 (peptides 113-167). Each pool contained 56 peptides, with each peptide at a concentration of 2 mg/ml. The bars represent the average number of ZEBOVGP-specific cytokine secreting splenocytes for 3 mice after subtracting the negative control well average. The assay was performed in triplicate, and the SEM is shown.
The T cell immunodominant epitope in ZEBOVGP is unknown. To further define the immunodominant epitopes the IFN-g ELISPOT assay was utilized using the peptides from pool # 2. All of the peptides from the second pool plus some neighbouring peptides were assigned to pools, numbered 1-16, based on a matrix configuration (Table 1). While each pool contained 8 peptides, each peptide was present in two pools. In order for a response to be considered positive for a specific peptide the signal had to be positive in both pools. There were very strong signals in pools 3, 6, 12, and 14. Using the matrix, peptides 72, 74, 96, and 98 were identified as possible immunodominant epitopes (Figure 2a). In order to confirm the results, the IFN-g ELISPOT assay was repeated with the individual peptides (Figure 2b). Peptide # 98 produced the strongest signal whereas peptide # 72 produced a much weaker signal, and the remaining neighbouring peptides produced background levels. Therefore the immunodominant epitope is peptide # 98 which contains the following amino acid sequence, HNTPVYKLDISEATQ and is found at amino acids #389-403 of GP.
Figure 2: ZEBOVGP Peptide Inducing an IFN-g Response. An IFN-g ELISPOT assay was performed in order to determine which ZEBOVGP peptide induced a ZEBOVGP-specific IFN-g response in VSV?G/ZEBOVGP immunized Balb/C mice. Splenocytes from VSV?G/ZEBOVGP immunized Balb/C mice were added to the IFN-g ELISPOT assay 14 days after the boost. The splenocytes were stimulated with (A) matrix peptide pools # 1-16 or (B) individual peptides (2 mg/ml). The bars represent the average number of ZEBOVGP-specific cytokine secreting splenocytes after subtracting the averaged negative controls for 6 and 2 mice, respectively. The assay was performed in triplicate, and the SEM is shown.
Murine protection study
[color=red]In order to determine whether the immunodominant HNTPVYKLDISEATQ peptide # 98 could induce a protective immune response, the peptide was used to immunize mice before a lethal high dose challenge with MA-ZEBOV (Table 2). As peptides alone generate poor immune responses the peptide was also used in conjunction with the carrier protein KLH, or Freund’s adjuvant[/color]. Groups of mice (n=5) were immunized subcutaneously with 50 mg of peptide, 3 times, 28 days apart before being challenged with 1000 LD50 MA-ZEBOV. A media only negative control group was used to demonstrate the lethality of the MA-ZEBOV infection. The VSV?G/ZEBOVGP (rVSV) vaccine given 28 days prior to challenge was used as a positive control to demonstrate complete protection against a high dose MA-ZEBOV infection [14]. All of the mice in the media only, peptide alone (PA), and peptide-KLH (PK) conjugated groups died by day 8. The mice in all 3 groups showed signs of illness as indicated by a significant drop in weight, along with an increased temperature on day 3 and 4 which decreased below normal shortly thereafter (Figure 3). However, while there was no difference in the mean time to death between the media and PA groups (5.7 and 5.5 days, respectively), the mice immunized with the PK had a mean time to death of 6.5 days which was significantly longer (p=0.016) than the media only group. The two groups of mice that were immunized with the peptide plus Freund’s adjuvant (PF) or peptide-KLH plus Freund’s adjuvant (PKF) demonstrated a 100% and 80% survival, respectively. There were no indications of illness in the PF, PKF or rVSV groups as indicated by the group average weight and temperatures (Figure 3). Even though the one mouse in the PKF group died, with a 7.5% drop in weight seen on day 6, all the other animals in the PKF group remained healthy, with no drop in weight, change in temperature, ruffled fur, or hunching. This demonstrates that peptide # 98 can induce a protective immune response in mice to such an extent that it can provide complete protection against a lethal high dose challenge of MA-EBOV.
Table 2: Immunodominant peptide # 98 protects against a MA-ZEBOV challenge in micea.aGroups of Balb/C mice (n=5) were immunized subcutaneously with media, 50µg peptide with (PF) or without Freund’s (PA), or 50 mg of peptide conjugated to KLH, with (PKF) or without Freund’s (PF) on days 0, 28, and 56. The positive control group VSV?G/ZEBOVGP (rVSV) were immunized intraperitoneally once on day 56 with 2X104 pfu/ml. The mice were challenged on day 84 with 1000 LD50 of MAZEBOV and their survival followed for 28 days. bData for all animals that died (number of animals that died are in parentheses). cSurvival rate on day 28 after challenge.dN/A: not applicable. eT test: compares mean time to death between the test group and media control fKaplan-Meier Survival Curve: compared the survival curves between the test group and media control.
Figure 3: MA-ZEBOV Post-challenge Weight and Temperature of Mice Vaccinated with Peptide # 98 Groups of Balb/C mice (n=5) were immunized with the various vaccines (PA= peptide alone, PK=peptide-KLH, PF=peptide + Freund’s, PKF=peptide-KLH + Freund’s) 3 times, 28 days apart. On the 28th day after the third immunization the mice were challenged with 1000 LD50 MA-ZEBOV. The weight and temperature of each mouse was taken daily after challenge for 2 weeks. The average weight for each group is shown for each day. The negative control mice received only medium. The positive control mice received the rVSV (VSV?G/ ZEBOVGP) vaccine, and were only immunized once and then challenged 28 days later.
As part of the protection study the humoural and T cell mediated immune responses were examined in the mice, leading up to the MA-ZEBOV challenge. An eVLP indirect ELISA was performed to determine the level of the ZEBOVGP-specific antibodies in the serum of the mice at day 0 (prebleed), and at 4, 8, and 11 weeks post-immunization (Figure 4A). As with the negative control mice that only received the medium, the PA group did not produce any ZEBOVGP-specific antibodies. When the peptide was bound to KLH (PK mice), a significant level of antibodies was seen by week 11. The addition of Freund’s adjuvant to either the peptide alone (PF) or the peptide-KLH conjugation (PKF) resulted in high antibody levels after only two immunizations. The positive control mice that received one injection of rVSV produced very high antibody titres. The levels of ZEBOVGP-specific antibodies in the PF and PKF groups were 57% and 74% respectively, after 2 immunizations, and 54% and 67% after 3 immunizations, of the titres seen with the rVSV positive control group. However, despite the higher titres in the PKF group one mouse did not survive whereas all mice survived in the PF group. When examining the titres in each mouse of the PKF group (Figure 4B), the mouse that did not survive (m1) had high background titres for the prebleed and the titres did not rise above background after immunization, suggesting an ineffective ZEBOVGP-specific humoural response to the immunization. Overall, immunization with peptide #98 did result in the production of ZEBOVGP-specific antibodies, but the strength of the antibody response was dependent upon the carrier molecules and adjuvant used.
Figure 4: ZEBOVGP-specific Antibody Titres in Mice Vaccinated with Peptide # 98. Groups of Balb/C mice (n=5) were immunized with the various vaccines (PA= peptide alone, PK=peptide-KLH, PF=peptide + Freund’s, PKF=peptide-KLH + Freund’s) 3 times, 28 days apart. (A) Serum taken from mice on the first day of immunization (prebleed), and then on weeks 4, 8, and 11 were tested for the presence of ZEBOVGP-specific antibodies in an eVLP indirect ELISA. A 1:100 dilution of the serum was used for all samples. The negative control mice received only medium. The positive control mice received the rVSV (VSV?G/ ZEBOVGP) vaccine, and were only immunized once, therefore only has a prebleed and 3 week serum sample. Each bar represents the average of the 5 mice in each group, with the error bars representing the standard deviation. The p values for the T test were determined by comparing the 4, 8, or 11 week sample to the prebleed within their own group, where *p=0.05, **p=0.005. (B) The prebleed, 4, 8, and 11 week ZEBOVGP-specific ELISA results are presented for each mouse (m1 to m5) in the PKF group. Mouse 1 (m1) is the animal that did not survive the MA-ZEBOV challenge.
In addition to the humoural response the ZEBOVGP-specific T cell mediated response was determined using an IFN-g and IL-2 ELISPOT assay (Figure 5). Two weeks after the third immunization, the spleens of 2 mice from each group were tested in order to determine the number of splenocytes responding to peptide # 98, or to two neighbouring peptides, # 97 and # 99. For all three peptides, there were no IFN-g or IL-2 secreting splenocytes in the medium, PA, or PK groups. The PF mice did not respond to any of the three peptides in the IFN-g assay, but did respond to peptide #98 in the IL-2 assay. In comparison the PKF group responded only to peptide #98 in both the IFN-g and IL-2 assay. The positive control rVSV immunized mice had a large number of splenocytes secreting IFN-g to peptide #98, and secreted IL-2 predominantly to peptide #98 with a much weaker response to peptides # 97 and # 99. Overall, the strongest T cell responses were seen in the groups receiving Freund’s adjuvant and they were highly specific for peptide # 98.
Figure 5: ZEBOV GP-specific Cytokine ELISPOT Response in Mice Vaccinated with Peptide # 98. Groups of Balb/C mice (n=2) were immunized with the various vaccines (PA= peptide alone, PK=peptide-KLH, PF=peptide+Freund’s, PKF=peptide-KLH+Freund’s) 3 times, 28 days apart. At two weeks after the third injection, an IFN-g and IL-2 ELISPOT assay was used to determine the number of ZEBOVGP-specific cytokine secreting splenocytes in the various groups of immunized mice. The splenocytes were stimulated with 2 mg/ml of either peptide # 97, # 98, or # 99. The negative control mice received only medium. The positive control mice received the rVSV (VSV?G/ZEBOVGP) vaccine, and were immunized once followed by a boost 28 days later, then 2 weeks later used in the assay. The bars represent the average number of ZEBOVGP-specific cytokine secreting splenocytes for the 2 mice in each group after subtracting the average of the negative control wells. The assay was performed in triplicate.
Discussion
A cytokine ELISPOT assay was used to determine the immunodominant epitope of the ZEBOV glycoprotein. As a reliable assay for determining memory cell immune responses in vaccine clinical trials, the ELISPOT assay is a sensitive and reproducible test for determining the antigen-specific T cell response at the single cell level. Determination of the frequency of the T cell response reflects the in vivo cellular frequency [15,16]. Additionally, the ELISPOT assay can be used to determine what cytokine profile is induced. In this study, Balb/C mice were immunized with the VSV?G/ZEBOVGP vaccine in which the VSV glycoprotein was replaced by the ZEBOV GP. Peptides spanning the ZEBOV GP were used to stimulate the splenocytes from these mice in 5 different cytokine ELISPOT assays in order to determine the ZEBOVGP-specific T cell cytokine profile. Upon exposure to the peptides the strongest responses were demonstrated in the Th1 cytokines IFN-g, TNF, and IL-2, whereas the two Th2 cytokine responses (IL- 4 and IL-10) were negligible. This suggests that ZEBOVGP presented in the context of VSV?G/ZEBOVGP during immunization produced ZEBOVGP-specific memory T cells secreting Th1 cytokines. This is an expected and typical immune response to viral infections. Of the three Th1 cytokines, the frequency of IFN-g producing splenocytes was 2.7 times higher than for IL-2 and TNF. Having a strong IFN-g response is vital for control of ZEBOV infections as ZEBOV has been shown to shut down IFN-g [17-19]. Therefore restoring a rapid IFN-g response early during an infection would be vital in countering the effects of a ZEBOV infection and tipping the scale towards survival.
IFN-g has strong antiviral effects that can inhibit viral replication directly, as well as stimulate host cellular immune response genes [20]. IFN-g is produced by memory and effector T cells but not naïve cells making the IFN-g ELISPOT assay a valuable tool for examining effector T cells responses in immunized animals [21]. In the VSV?G/ ZEBOVGP immunized mice a higher percentage of ZEBOVGPspecific splenocytes were secreting IFN-g therefore the IFN-g ELISPOT assay was used to determine the immunodominant ZEBOVGP epitope. The frequency of ZEBOVGP-specific splenocytes did not span the entire region of GP. The initial cytokine profile assays demonstrated that ZEBOVGP peptide pool # 2 induced overwhelmingly the highest number of spot forming units (SFU) for all three Th1 cytokine secreting splenocytes. To further define the immunodominant epitope, the peptides from pool # 2 were split into another set of pools according to a matrix in order to determine which specific peptides were inducing the IFN-g T cell response. Of the four possible peptides identified (# 72, 74, 96, and 98) only peptide # 98 stimulated a strong IFN-g response suggesting peptide #98 was the immunodominant epitope. This peptide HNTPVYKLDISEATQ (amino acids 389-403), which is conserved across both the Mayinga and Kikwit strains, is located in the mucin domain of GP. The mucin domain is believed to be responsible for the cytopathicity in GP expressing cells lines, and may play a critical role in the pathogenesis of filoviral disease [22-24]. Interestingly, protective monoclonal antibody 6D8-1-2 also binds this epitope which suggests that this epitope may elicit both a humoural and a cellular immune response [25].
Finding a strongly immunogenic epitope in a protein does not guarantee that using the peptide in a vaccine can prevent disease. Therefore, in order to demonstrate the capacity of peptide # 98 to induce protective immunity, mice were immunized with the peptide alone (PA), peptide conjugated to KLH (PK), or peptide along with Freund’s adjuvant (PF and PKF). Small peptides and proteins are not very effective at eliciting a strong immune response therefore it is not unexpected that peptide alone did not increase survival from a lethal MA-ZEBOV infection. Therefore, KLH was employed as a carrier for the peptide in order to make the peptide more immunogenic [26]. While the KLH-conjugated peptide (PK) increased the mean time to death it did not prevent death. However, this model used a high dose challenge and it is possible that there could have been an improved rate of survival with a lower challenge dose of MA-ZEBOV. A more potent mechanism for enhancing the immune response is to inject the peptide in the presence of an adjuvant such as Freund’s [27]. The adjuvanted peptide was extremely effective as it increased survival to 100%. This demonstrates that peptide #98 induces a protective immune response.
The humoural and T cell mediated immune response elicited by peptide #98 was compared for each of the various vaccine protocols. The medium negative control, and the peptide alone (PA) immunization did not stimulate any immune response. This is not surprising as small peptides are very weak immunogens. One possible reason is that the peptide vaccine contains just one epitope and therefore may induce only a very narrow and weak response. Or, peptides in aqueous solutions may induce tolerance as the peptide does not contain “danger signals” that are required to induce inflammatory responses needed to stimulate the immune system [28]. Therefore, an adjuvant or carrier is often required to induce this response (reviewed in [29]. Adding the carrier protein KLH to the peptide (PK) resulted in a weak ZEBOVGPspecific antibody response at week 11 but no noticeable T cell response. This is reflected in the PK vaccines ability to increase the mean time to death but not improve survival. The addition of Freund’s adjuvant to the peptide (PF and PKF) provided the necessary signals which resulted in a strong ZEBOVGP-specific antibody response after just two immunizations; and splenocytes secreting IL-2 (PKF and PF) or IFN-g (PKF) after 3 immunizations. The T and B cell response is not as strong as what is observed in mice immunized with the VSV?G/ ZEBOVGP vaccine, most likely due to several reasons. Firstly, the antigenic dose of the VSV?G/ZEBOVGP vaccine would be higher as it is a live replicating virus. Secondly, as VSV?G/ZEBOVGP includes the entire ZEBOV GP, it contains more B and T cell epitopes than the peptide vaccine, and would thus provide a broader antigenic stimulus. This is reflected in the IL-2 ELISPOT results where there were IL-2 secreting splenocytes specific for peptide # 98 as well as peptides # 97 and # 99. Finally, the adjuvants “danger signals” provided by VSV?G/ ZEBOVGP will be quantitatively and qualitatively different than those provided by Freund’s. Surprisingly, the PKF group which had a stronger B- and T- cell response than the PK group, had one mouse (m1) die. This mouse was later found to have a weak/absent ZEBOVGP-specific antibody response, as the antibody titres never exceeded the initially high background levels. There are no corresponding ELISPOT results for this mouse as the ELISPOT results come from mice not included in the challenge. However, previous work has shown that CD8+ T cells are not required for survival yet passive transfer of ZEBOVGP-specific antibodies does provide protection [14]. Therefore, it is possible that the one mouse that died did not have a sufficient antibody response against the ZEBOV GP, for reasons unknown. The PF group which did not have any IFN-g secreting splenocytes did have complete survival. This evidence supports the necessity for antibodies in promoting survival during a ZEBOV infection.
The peptide sequence HNTPVYKLDISEATQ is conserved across the Mayinga and Kikwit strains ofZaire Ebolavirus, but is not conserved across the various species of Ebola. Therefore this peptide may not provide cross-protection against other Ebola virus species. The peptide HNTPVYKLDISEATQ is not a previously identified CD8+ CTL epitope. Two other H-2d restricted peptides that have been identified in Balb/C mice include VSTGTGPGAGDFAFHK (amino acids 141-155) identified using the Venezuelan Equine Encephalitis Virus Replicon (VRP) expressing the Zaire glycoprotein as a vaccine [30], and the LYDRLASTI (amino acids 161-169) identified using the liposome plus irradiated Ebola virus vaccine (L(EV)) [31]. Both of these studies immunized with vaccines containing the complete glycoprotein and then either identified the immunodominant CTL epitope using overlapping peptides, or were tested against peptides predicted by software to be strong binders for H-2d, respectively. Possible reasons why each study did not pick up the others immunodominant peptides may be due to the fact that all three approaches used different assays to identify the peptides, as well as different vaccine delivery systems. In the Olinger VRP-GP vaccine study, the VSTGTGPGAGDFAFHK epitope was identified using intracellular cytokine staining (ICS) by flow cytometry, yet the epitope was undetected in their IFN-g ELISPOT assay. This is consistent with our results where we also did not detect their epitope in our IFN-g ELISPOT assay, and we did not perform an ICS assay. The L(EV) vaccine used a software to determine the binding predictions for H-2d based on the amino acid sequence of ZEBOV GP. Because their predicted peptide sequence did not match that of our peptide # 98, our peptide was not part of their ELISPOT, or Cr51 CTL assay. We however, did not identify their predicted epitope in our ELISPOT. This may be because using a peptide matrix pool where there are 8 peptides in one pool, may result in the response to one peptide being overwhelmed by the response to another peptide. Comparison of the various assays to define immunodominant epitopes needs to be addressed to determine which method might predict the most effective epitope.
It is difficult to say which epitope will be most effective in providing protection in an in vivo model. However, the current study is the first to demonstrate that the 15 amino acid peptide based on the identified epitope can provide protection in a mouse model. The other two studies identified their epitopes as either CD4+ or CD8+ specific. In our study the ELISPOT assay was not able to differentiate between CD4+ or CD8+ T lymphocytes, therefore we used a web based epitope prediction software (IEBC Analysis Resource at http://tools.immuneepitope.org/main/) and identified epitopes within HNTPVYKLDISEATQ that bound to MHCI and MHCII with an IC50 of approximately 30 mM, and 4-7 mM, respectively. This suggests that the peptide has a 4-8 times higher binding affinity for MHCII than MHCI. Activation of CD4+ T lymphocytes would suggest the activation of a humoural immune response. Interestingly, our peptide was also found to be bound by monoclonal antibody 6D8-1-2 [25].The identification of distinctly different epitopes using 3 different vaccine platforms may be due to differences in the route of immunization as well as the vaccine formulation. The L(EV) vaccine generated a CTL response, but the irradiated EV in absence of the liposome did not, suggesting that the additional signals, or route of cellular entry provided by the liposome can influence the immune response. Liposome encapsulated antigen is presumably processed by a different intracellular pathway which could possibly lead to a different immune response [32]. The danger signals presented by the other components of each vaccine would also be distinctly different which may manifest in different cytokines being secreted or different cells being activated. Additional research into the protective mechanisms has to be determined for vaccines and Ebola virus infections.
All of the data taken together suggests that peptide # 98 can induce both a T and B cell immune response that is sufficient to protect against a high dose lethal challenge of MA-ZEBOV. The level of induction of the B and T cells is not as strong as what is observed with the rVSV vaccine. However, the peptide contains only one epitope whereas the rVSV vaccine contains numerous epitopes. The fact that a single peptide can provide sufficient protection suggests that with a suitable delivery system and adjuvant, a subunit vaccine containing the single peptide could be an effective vaccine that is safer than the live replicating virus vaccine. Although the antibody titres were high there is evidence that Th1 cell mediated immune response may also play an important role in providing protection against a ZEBOV infection. The cytokine profile suggested a Th1 cytokine response, and since the IFN-g ELISPOT was used to determine the immunodominant epitope it is plausible that the cell mediated immune response is also a factor. The ELISPOT assay used here can only quantitate an overall T cell response, and does not determine whether the ZEBOVGP-specific response was mediated by CD4+ or CD8+ T cells. Future experiments should include a flow cytometry based intracellular cytokine assay that can differentiate which T lymphocyte recognizes the immunodominant epitope # 98 identified here. Additionally, further investigation in to the relative contribution of the T and B cell response towards protection by peptide # 98 could be determined through the adoptive T cell or antibody transfer studies, or through the vaccination of T- or B-cell deficient mice. Identifying the cytokine profile and immunodominant ZEBOVGP epitope in the VSV?G/ZEBOVGP vaccine aids in determining the correlates of immune protection against ZEBOV, and will play an important role in developing assays to establish the efficacy of the vaccine in eliciting a protective immune response in vaccinated individuals. The VSV?G/ ZEBOVGP vaccine completely protects non-human primates against a lethal high dose challenge indicating that GP is sufficient to elicit protection [1-3]. While using VSV as the vector carrying ZEBOVGP to immunize against a ZEBOV infection, the identification of the cytokine profile and immunodominant epitope will also allow for the development of future subunit vaccines using peptide #98.
Acknowledgements
We would like to thank the Veterinary Technical Services at the National Microbiology Laboratory in Winnipeg for their animal work, and Jonathan Audet for his help with the manuscript. The source of funding for this project is from the Chemical, Biological, Radiological-nuclear, and Explosives Research and Technology Inititative (CRTI), and the Public Health Agency of Canada (PHAC).
Has anyone listened/watched the Rodman NY presentation besides me? I just accessed it again from the company website. If you have not seen/heard the presentation, you should. Oakes paints a far different picture than the article published by an obvious shorter. You go to their press releases, and click on the 9/2 press release, it then opens to a window where you see links to see/hear the presentation. You may have to play around with it to get sound and the power point at the same time. I had to use the flash player to hear it, you might have to use windows media or another player selection tab at the bottom of the screen. This presentation was in NY city in a conference where the keynote speaker was Colin Powell. It was attended by some of the brightness minds on Wall St. I would guess it also had some US SEC etc. regulators in attendance. It is hard for me to envision someone outright lying in that environment, the PP shows they are in process of uplisting to Major US Stock exchange and much more that seems to be almost 180 degrees from the Monday's SA article.
Would appreciate thoughts of others if you are able to access it.
Thanks
Last's week Presentation:
The presentation is available to public on the link in the previous post, you may have to still sign up. It is also available on co.web site. It is tricky to get both audio and the 22 page power point as it depends which OS you are using and which version of chrome or IE etc. I use Windows 7 and used IE and had to use the Flash player selection at the bottom, windows media player did not work.
It is worth the effort if you are interested in this stock. 20 min Presentation by CEO with a little Q and A at the end. The PP presentation is 22 pages and very informative. The most notable things to me were he again mentioned ongoing efforts to uplist. Also, in the ongoing trial studies, if any of them are successful, most would generate tens of $million per year in revenue but if the Rhu Arthritis vaccine is successful, it would potentially generate 100's of $millions per year in revenue. Also, Big Pharma will know prior to releasing the final results and approvals by various FDA's worldwide their chances and if things are looking good would probably want to lock up a supply contract in advance since all big pharma know that the KLH supply is quite limited and it is just a matter of time before Natural Harvesting will be banned to protect the already rare species. If 3 or 4 trials are successful, probably would be a supply shortage, at least at first. Also, he said burn rate is about $500k a month and they can run through 2015 with current cash on hand with no new revenue.