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The Abyss

The Abyss is a 1989 American science fiction film written and directed by James Cameron and starring Ed Harris, Mary Elizabeth Mastrantonio, and Michael Biehn. When an American submarine sinks in the Caribbean, a US search and recovery team works with an oil platform crew, racing against Soviet vessels to recover the boat. Deep in the ocean, they encounter something unexpected.

The film was released on August 9, 1989, receiving generally positive reviews and grossed $90 million. It was nominated for four Academy Awards and won the Academy Award for Best Visual Effects.

Plot
In January 1994, the U.S. Ohio-class submarine USS Montana has an encounter with an unidentified submerged object and sinks near the Cayman Trough. With Soviet ships moving in to try to salvage the sub and a hurricane moving over the area, the U.S. government sends a SEAL team to Deep Core, a privately owned experimental underwater drilling platform near the Cayman Trough, to use it as a base of operations. The platform's designer, Dr. Lindsey Brigman, insists on going along with the SEAL team, even though her estranged husband Virgil "Bud" Brigman is the current foreman.

During the initial investigation of Montana, a power cut in the team's submersibles leads to Lindsey seeing a strange light circling the sub, which she later calls a "non-terrestrial intelligence" or "NTI". Lt. Hiram Coffey, the SEAL team leader, is ordered to accelerate their mission and takes one of the mini-subs without Deep Core's permission to recover a Trident missile warhead from Montana just as the storm hits above, leaving the crew unable to disconnect from their surface support ship in time. The cable crane is torn from the ship and falls into the trench, dragging Deep Core to the edge before it stops. The rig is partially flooded, killing several crew members and damaging its power systems.

The crew waits out the storm so they can restore communications and be rescued. As they struggle against the cold, they find the NTIs have formed an animated column of water to explore the rig, which they equate to an alien version of a remotely operated vehicle. Though they treat it with curiosity, Coffey is agitated and cuts it in half by closing a pressure bulkhead on it, causing it to retreat. Realizing that Coffey is suffering paranoia from high-pressure nervous syndrome, the crew spies on him through an ROV, finding him and another SEAL arming the warhead to attack the NTIs. To try to stop him, Bud fights Coffey, but Coffey escapes in a mini-sub with the primed warhead. Bud and Lindsey give chase in the other sub, damaging both. Coffey is able to launch the warhead into the trench, but his sub drifts over the edge and implodes from the pressure, killing him. Bud's mini-sub is inoperable and taking on water. With only one functional diving suit, Lindsey opts to enter deep hypothermia and trigger her mammalian diving reflex when the ocean's cold water engulfs her. Bud swims back to the platform with her body; there, he and the crew use a defibrillator and administer CPR, and they revive her.

It is decided that they need to disarm the warhead, which is more than 2 miles below them. One SEAL, Ensign Monk, helps Bud use an experimental diving suit equipped with a liquid breathing apparatus to survive to that depth, though he will only be able to communicate through a keypad on the suit. Bud begins his dive, assisted by Lindsey's voice to keep him coherent against the effects of the mounting pressure, and he reaches the warhead. Monk guides him in successfully disarming it. With little oxygen left in the system, Bud explains that he knew it was a one-way trip, and he tells Lindsey he loves her. As he waits for death, an NTI approaches Bud, takes his hand, and guides him to a massive alien city deep in the trench. Inside, the NTIs create an atmospheric pocket for Bud, allowing him to breathe normally. The NTIs then play back Bud's message to his wife and look at each other with understanding.

On Deep Core, the crew is waiting for rescue when they see a message from Bud that he met some friends and warns them to hold on. The base shakes, and lights from the trench herald the arrival of the alien ship. It rises to the ocean's surface, with Deep Core and several of the surface ships run aground on its hull. The crew of Deep Core exits the platform, surprised they are not dead from the sudden decompression. They see Bud walking out of the alien ship, and Lindsey races to hug him.

Special Edition
In the extended version, the events in the film are played against a backdrop of conflict between the United States and the Soviet Union, with the potential for all-out war. The sinking of Montana additionally fuels the aggression. There is additionally more conflict between Bud and Lindsey in regard to their former relationship. The primary addition is the ending: When Bud is taken to the alien ship, the aliens begin by showing him images of war and aggression from news sources around the globe. The aliens then create massive megatsunamis that threaten the world's coasts, but stop them short before they hit. Bud asks why they spared the humans, and they show Bud his message to Lindsey before bringing him, the alien ship, and Deep Core to the surface.

Production
H. G. Wells was the first to introduce the notion of a sea alien in his 1897 short story "In the Abyss". The idea for The Abyss came to James Cameron when, at age 17 and in high school, he attended a science lecture about deep sea diving by a man, Francis J. Falejczyk, who was the first human to breathe liquid through his lungs in experiments conducted by Johannes A. Kylstra at Duke University. He subsequently wrote a short story that focused on a group of scientists in a laboratory at the bottom of the ocean. The basic idea did not change, but many of the details were modified over the years. Once Cameron arrived in Hollywood, he quickly realized that a group of scientists was not that commercial and changed it to a group of blue-collar workers. While making Aliens, Cameron saw a National Geographic film about remote operated vehicles operating deep in the North Atlantic Ocean. These images reminded him of his short story. He and producer Gale Anne Hurd decided that The Abyss would be their next film. Cameron wrote a treatment combined with elements of a shooting script, which generated a lot of interest in Hollywood. He then wrote the script, basing the character of Lindsey on Hurd and finished it by the end of 1987. Cameron and Hurd were married before The Abyss, separated during pre-production, and divorced in February 1989, two months after principal photography.

Pre-production
The cast and crew trained for underwater diving for one week in the Cayman Islands. This was necessary because 40% of all live-action principal photography took place underwater. Furthermore, Cameron's production company had to design and build experimental equipment and develop a state-of-the-art communications system that allowed the director to talk underwater to the actors and dialogue to be recorded directly onto tape for the first time.

Cameron had originally planned to shoot on location in the Bahamas where the story was set but quickly realized that he needed to have a completely controlled environment because of the stunts and special visual effects involved. He considered shooting the film in Malta, which had the largest unfiltered tank of water, but it was not adequate for Cameron's needs. Underwater sequences for the film were shot at a unit of the Gaffney Studios, situated south of Cherokee Falls, outside Gaffney, South Carolina, which had been abandoned by Duke Power officials after previously spending $700 million constructing the Cherokee Nuclear Power Plant, along Owensby Street, Gaffney, South Carolina.

Two specially constructed tanks were used. The first one, based on the abandoned plant's primary reactor containment vessel, held 7.5 e6USgal of water, was 55 feet (18 m) deep and 209 feet (70 m) across. At the time, it was the largest fresh-water filtered tank in the world. Additional scenes were shot in the second tank, an unused turbine pit, which held 2.5 e6USgal of water. As the production crew rushed to finish painting the main tank, millions of gallons of water poured in and took five days to fill. The Deepcore rig was anchored to a 90-ton concrete column at the bottom of the large tank. It consisted of six partial and complete modules that took over half a year to plan and build from scratch.

Can-Dive Services Ltd., a Canadian commercial diving company that specialized in saturation diving systems and underwater technology, specially manufactured the two working craft (Flatbed and Cab One) for the film. Two million dollars was spent on set construction.

Filming was also done at the largest underground lake in the world—a mine in Bonne Terre, Missouri, which was the background for several underwater shots.

Principal photography
The main tank was not ready in time for the first day of principal photography. Cameron delayed filming for a week and pushed the smaller tank's schedule forward, demanding that it be ready weeks ahead of schedule. Filming eventually began on August 15, 1988, but there were still problems. On the first day of shooting in the main water tank, it sprang a leak and 150,000 USgal of water a minute rushed out. The studio brought in dam-repair experts to seal it. In addition, enormous pipes with elbow fittings had been improperly installed. There was so much water pressure in them that the elbows blew off.

Cameron's cinematographer, Mikael Salomon, used three cameras in watertight housings that were specially designed. Another special housing was designed for scenes that went from above-water dialogue to below-water dialogue. The filmmakers had to figure out how to keep the water clear enough to shoot and dark enough to look realistic at 2,000 feet (700 m), which was achieved by floating a thick layer of plastic beads in the water and covering the top of the tank with an enormous tarpaulin. Cameron wanted to see the actors' faces and hear their dialogue, and thus hired Western Space and Marine to engineer helmets which would remain optically clear underwater and installed state-of-the-art aircraft quality microphones into each helmet. Safety conditions were also a major factor with the installation of a decompression chamber on site, along with a diving bell and a safety diver for each actor.

The breathing fluid used in the film actually exists but has only been thoroughly investigated in animals. Over the previous 20 years it had been tested on several animals, who survived. The rat shown in the film was actually breathing liquid and survived unharmed. Production consulted with Dr. Kylstra on the proper use of the breathing fluid for the film. Ed Harris did not actually breathe the fluid. He held his breath inside a helmet full of liquid while being towed 30 feet (10 m) below the surface of the large tank. He recalled that the worst moments were being towed with fluid rushing up his nose and his eyes swelling up.

Actors played their scenes at 33 feet (11 m), too shallow a depth for them to need decompression, and rarely stayed down for more than an hour at a time. Cameron and the 26-person underwater diving crew sank to 50 feet (17 m) and stayed down for five hours at a time. To avoid decompression sickness, they would have to hang from hoses halfway up the tank for as long as two hours, breathing pure oxygen.

The cast and crew endured over six months of grueling six-day, 70-hour weeks on an isolated set. At one point, Mary Elizabeth Mastrantonio had a physical and emotional breakdown on the set and on another occasion, Ed Harris burst into spontaneous sobbing while driving home. Cameron himself admitted, "I knew this was going to be a hard shoot, but even I had no idea just how hard. I don't ever want to go through this again".

For example, for the scene where portions of the rig are flooded with water, he realized that he initially did not know how to minimize the sequence's inherent danger. It took him more than four hours to set up the shot safely. Actor Leo Burmester said, "Shooting The Abyss has been the hardest thing I've ever done. Jim Cameron is the type of director who pushes you to the edge, but he doesn't make you do anything he wouldn't do himself." A lightning storm caused a 200-foot (65 m) tear in the black tarpaulin covering the main tank. Repairing it would have taken too much time, so the production began shooting at night. In addition, blooming algae often reduced visibility to 20 feet (6 m) within hours. Over-chlorination led to divers' skin burning and exposed hair being stripped off or turning white.

As production went on, the slow pace and daily mental and physical strain of filming began to wear on the cast and crew. Mary Elizabeth Mastrantonio remembered, "We never started and finished any one scene in any one day". At one point, Cameron told the actors to relieve themselves in their wetsuits to save time between takes. While filming one of many takes of Mastrantonio's character's resuscitation scene—in which she was soaking wet, topless and repeatedly being slapped and pounded on the chest—the camera ran out of film, prompting Mastrantonio to storm off the set yelling, "We are not animals!"

For some shots in the scene that focus on Ed Harris, he was yelling at thin air because Mastrantonio refused to film the scene again. Michael Biehn also grew frustrated by the waiting. He claimed that he was in South Carolina for five months and only acted for three to four weeks. He remembered one day being ten meters underwater and "suddenly the lights went out. It was so black I couldn't see my hand. I couldn't surface. I realized I might not get out of there." Harris recalled: "One day we were all in our dressing rooms and people began throwing couches out the windows and smashing the walls. We just had to get our frustrations out."

Cameron responded to these complaints, saying, "For every hour they spent trying to figure out what magazine to read, we spent an hour at the bottom of the tank breathing compressed air." After 140 days and $4 million over budget, filming finally wrapped on December 8, 1988. Before the film's release, there were reports from South Carolina that Ed Harris was so upset by the physical demands of the film and Cameron's dictatorial directing style that he said he would refuse to help promote the motion picture. Harris later denied this rumor and helped promote the film. However, after its release and initial promotion, Harris publicly disowned the film, saying "I'm never talking about it and never will." Mary Elizabeth Mastrantonio also disowned the film, saying, "The Abyss was a lot of things. Fun to make is not one of them."

Post-production
To create the alien water tentacle, Cameron initially considered cel animation or a tentacle sculpted in clay and then animated via stop-motion techniques with water reflections projected onto it. Phil Tippett suggested Cameron contact Industrial Light & Magic. The special visual effects work was divided up among seven FX divisions with motion control work by Dream Quest Images and computer graphics and opticals by ILM. ILM designed a program to produce surface waves of differing sizes and kinetic properties for the pseudopod, which was referred to by ILM informally as the "water weenie." For the moment where it mimics Bud and Lindsey's faces, Ed Harris had eight of his facial expressions scanned while twelve of Mastrantonio's were scanned via software used to create CGI sculptures. The set was photographed from every angle and digitally recreated so that the pseudopod could be accurately composited into the live-action footage. For the sequence where Mary Elizabeth Mastrantonio touches the surface of the pseudopod, then-ILM receptionist Alia Agha stood in as a hand model. The company spent six months to create 75 seconds of computer graphics needed for the creature. An optical printer was used for the compositing of the sequence, except from the shot where the pseudopod turns back into ordinary water, which was composited digitally. The film was to have opened on July 4, 1989, but its release was delayed for more than a month by production and visual effects problems. The CGI-animated sequences were supervised by ILM animation director Wes Takahashi.

Studio executives were nervous about the film's commercial prospects when preview audiences laughed at scenes of serious intent. Industry insiders said that the release delay was because nervous executives ordered the film's ending completely re-shot. There was also the question of the size of the film's budget: 20th Century Fox stated that the budget was $43 million, a figure Cameron himself has reiterated. However, estimates put the figure higher with The New York Times estimating the cost at $45 million and one executive claiming it cost $47 million, while box office revenue tracker website The Numbers lists the production budget at $70 million.

Box office
The Abyss was released on August 9, 1989, in 1,533 theaters, where it grossed $9.3 million on its opening weekend and ranked #2 at the box office behind Parenthood. It went on to make $54.2 million in North America and $35.5 million throughout the rest of the world for a worldwide total of $89.8 million.

Critical response
On Rotten Tomatoes, a review aggregator, The Abyss has an 89% approval rating based on 55 reviews and an average rating of 7.40/10. The critical consensus states: "The utterly gorgeous special effects frequently overshadow the fact that The Abyss is also a totally gripping, claustrophobic thriller, complete with an interesting crew of characters." On Metacritic, the film has an average score of 62 out of 100, based on 14 critics indicating "generally favorable reviews". The reviews tallied therein are for both the theatrical release and the Special Edition. Audiences polled by CinemaScore gave the film an average grade of "A-" on an A+ to F scale.

David Ansen of Newsweek, summarizing the theatrical release, wrote, "The payoff to The Abyss is pretty damn silly — a portentous deus ex machina that leaves too many questions unanswered and evokes too many other films." In her review for The New York Times, Caryn James wrote that the film had "at least four endings," and "by the time the last ending of this two-and-a-quarter-hour film comes along, the effect is like getting off a demon roller coaster that has kept racing several laps after you were ready to get off." Chris Dafoe, in his review for The Globe and Mail, wrote, "At its best, The Abyss offers a harrowing, thrilling journey through inky waters and high tension. In the end, however, this torpedo turns out to be a dud—it swerves at the last minute, missing its target and exploding ineffectually in a flash of fantasy and fairy-tale schtick." While praising the film's first two hours as "compelling", the Toronto Star remarked, "But when Cameron takes the adventure to the next step, deep into the heart of fantasy, it all becomes one great big deja boo. If we are to believe what Cameron finds way down there, E.T. didn't really phone home, he went surfing and fell off his board." Mike Clark of USA Today gave the film three out of four stars and wrote, "Most of this underwater blockbuster is 'good,' and at least two action set pieces are great. But the dopey wrap-up sinks the rest 20,000 leagues."

In her review for The Washington Post, Rita Kempley wrote that the film "asks us to believe that the drowned return to life, that the comatose come to the rescue, that driven women become doting wives, that Neptune cares about landlubbers. I'd sooner believe that Moby Dick could swim up the drainpipe." Halliwell's Film Guide stated the film was, "despite some clever special effects, a tedious, overlong fantasy that is more excited by machinery than people." Conversely, Peter Travers of Rolling Stone enthused, "[The Abyss is] the greatest underwater adventure ever filmed, the most consistently enthralling of the summer blockbusters…one of the best pictures of the year." John Ferguson of Radio Times awarded it three stars out of five, stating "For some, this was James Cameron's Waterworld, a bloated, sentimental epic from the king of hi-tech thrillers. Some of the criticism was deserved, but it remains a fascinating folly, a spectacular and often thrilling voyage to the bottom of the sea [...] Cameron excels in cranking up the tension within the cramped quarters and the effects are awe-inspiring and deservedly won an Oscar. It's only marred by being overlong and by its sentimental attachment to aliens."

The release of the Special Edition in 1993 garnered much praise. Each giving it thumbs up, Gene Siskel remarked, "The Abyss has been improved," and Roger Ebert added, "It makes the film seem more well rounded." In the book Reel Views 2, James Berardinelli comments, "James Cameron's The Abyss may be the most extreme example of an available movie that demonstrates how the vision of a director, once fully realized on screen, can transform a good motion picture into a great one."

Accolades
The Abyss won the 1990 Oscar for Best Visual Effects (John Bruno, Dennis Muren, Hoyt Yeatman, and Dennis Skotak). It was also nominated for:
 * Best Art Direction (Art Direction: Leslie Dilley; Set Decoration: Anne Kuljian)
 * Best Cinematography (Mikael Salomon)
 * Best Sound (Don J. Bassman, Kevin F. Cleary, Richard Overton and Lee Orloff)

Many other film organizations, such as the Academy of Science Fiction, Fantasy & Horror Films, and the American Society of Cinematographers, also nominated The Abyss. The film ended up winning a total of three other awards from these organizations.

Soundtrack
The soundtrack to The Abyss was written by Alan Silvestri and released by Varèse Sarabande on August 22, 1989. In 2014, they issued a limited-edition (3,000 copies), two-disc album featuring the complete score minus the end credits medley, which is absent from both releases.

Special Edition
Rumors circulated from the film's opening weeks of sequences cut from the film's third act. Pressure to cut the film's running time stemmed from both distribution concerns and Industrial Light & Magic's then-inability to complete the required sequences. The looming three-hour length also limited the number of times the film could be shown each day, though Dances with Wolves (1990) would challenge industry notions. Test audience screenings revealed mixed reactions to the sequences as they appeared in their unfinished form.

Cameron held final cut provided that the film met a running time of roughly two hours and 15 minutes. He later noted: "Ironically, the studio brass were horrified when I said I was cutting the wave."

What emerges in the winnowing process is only the best stuff. And I think the overall caliber of the film is improved by that. I cut only two minutes of Terminator. On Aliens, we took out much more. I even reconstituted some of that in a special (TV) release version.

The sense of something being missing on Aliens was greater for me than on The Abyss, where the film just got consistently better as the cut got along. The film must function as a dramatic, organic whole. When I cut the film together, things that read well on paper, on a conceptual level, didn't necessarily translate to the screen as well. I felt I was losing something by breaking my focus. Breaking the story's focus and coming off the main characters was a far greater detriment to the film than what was gained. The film keeps the same message intact at a thematic level, not at a really overt level, by working in a symbolic way.

Cameron elected to remove the wave sequences along with other, shorter scenes elsewhere in the film, reducing the running time from roughly two hours and 50 minutes to two hours and 20 minutes, and diminishing his signature themes of nuclear peril and disarmament. Subsequent test audience screenings drew substantially better reactions.

Mary Elizabeth Mastrantonio publicly expressed regret about some of the scenes selected for removal from the film's theatrical cut, saying: "There were some beautiful scenes that were taken out. I just wish we hadn't shot so much that isn't in the film."

Shortly after the film's premiere, Cameron and video editor Ed Marsh created a longer video cut of The Abyss for their own use that incorporated dailies. With the tremendous success of Cameron's Terminator 2: Judgment Day in 1991, Lightstorm Entertainment secured a five-year, $500 million financing deal with 20th Century Fox for films produced, directed or written by Cameron. The contract allocated roughly $500,000 of the amount to complete The Abyss. ILM was commissioned to finish the work they had started three years earlier, with many of the same people who had worked on it originally.

The CGI tools developed for Terminator 2: Judgment Day allowed ILM to complete the tidal wave sequence, as well as correcting flaws in rendering for all their other work done for the film.

The tidal wave sequence had originally been designed by ILM as a physical effect, using a plastic wave, but Cameron was dissatisfied with the end result, and the sequence was scrapped. By the time Cameron was ready to revisit The Abyss, ILM's CGI prowess had finally progressed to an appropriate level, and the wave was rendered as a CGI effect. Terminator 2: Judgment Day screenwriter and frequent Cameron collaborator William Wisher had a cameo in the scene as a reporter in Santa Monica who catches the first tidal wave on camera.

When it was discovered that original production sound recordings had been lost, new dialogue and foley were recorded, but since Kidd Brewer had died before he could return to re-loop his dialog, producers and editors had to lift his original dialogue tracks from the remaining optical-sound prints of the dailies. The Special Edition was therefore dedicated to his memory as a result.

As Alan Silvestri was not available to compose new music for the restored scenes, Robert Garrett, who had composed temporary music for the film's initial cutting in 1989, was chosen to create new music. The Special Edition was completed in December 1992, with 28 minutes added to the film, and saw a limited theatrical release in New York City and Los Angeles on February 26, 1993, and expanded to key cities nationwide in the following weeks. Both versions of the film continue to receive public exhibitions, including a screening of an original 35mm print of the theatrical cut on August 20, 2019, in New York City.

On November 13, 2023, Cameron announced in a video message via Twitter that a 4K remastered transfer of The Abyss: Special Edition will return to theaters for a one-night-only event on December 6, 2023; 20th Century Studios released a new trailer the same day to hype up the screening. Cameron explained: "If you haven't seen the film before, this is the way to experience it and if you have, you'll be seeing the film I actually set out to make, with some big surprises not seen in the originally released version. I hope you'll take advantage of seeing The Abyss, my first ocean film, back in theaters."

Home media
The first THX-certified LaserDisc title of the Special Edition Box Set was released in April 1993, in both widescreen and full-screen formats, and it was a best-seller for the rest of the year. The Special Edition was released on VHS on August 20, 1996, as a part of Fox Video's Widescreen Series, with a seven-minute behind-the-scenes featurette with footage that did not appear in the Under Pressure: The Making of The Abyss documentary that was included on the Laserdisc and DVD releases. The film was released on DVD in 2000 and contains both the theatrical (145 minutes) and Special Edition (171 minutes) versions of the film via seamless branching along with—on a second disc—the Laserdisc's extensive text, artwork and photographic documentation of the film's production, a ten-minute featurette, and the sixty-minute documentary Under Pressure: The Making of The Abyss. The second disc was removed in subsequent reprints.

In 2014, the pay cable channels Cinemax and HBO began broadcasting both versions of the film in 1080p. Netflix's UK service began offering the theatrical version in 1080p in 2017. At an October, 2014 event James Cameron and Gale Anne Hurd were asked about a future Blu-ray release for the film. Cameron gestured to the head of Fox Home Entertainment, implying the decision lay with the studio. Five months later, another article suggested a spat between Cameron and 20th Century Fox Home Entertainment was responsible for the delay. While promoting the upcoming 30th-anniversary Blu-ray release of Aliens at Comic-Con in San Diego in July 2016, James Cameron confirmed that he was working on a remastered 4K transfer of The Abyss and that it would be released on Blu-ray for the first time in early 2017. Cameron added, "We've done a wet-gate 4K scan of the original negative, and it's going to look insanely good. We're going to do an authoring pass in the DI for Blu-ray and HDR at the same time."

In March 2019, digital intermediate colorist Skip Kimball posted a photo to his Instagram suggesting that he was working on the film. In November 2018, Cameron told Empire magazine that a Blu-ray transfer was "complete for my review" and he hoped it would be ready before 2019.

In December 2021, during a promotional interview for the new book Tech Noir: The Art of James Cameron with website Space.com, director Cameron made the following statement: "Yeah, we finished the transfer and I wanted to do it myself because Mikael [Salomon] did such a beautiful job with the cinematography on that film. It is truly, truly gorgeous cinematography. That was before I started to assert myself in terms of lighting and asking the cinematographer to do certain things. I'd compose with the camera and choose the lenses, but I left the lighting to him. He did a remarkable job on that movie that I appreciate better now than I did even as we were making it ... So I just recently finished the high-def transfer a couple of months ago[,] so presumably there’ll be Blu-rays and it will stream with a proper transfer from now on. I appreciate what you said about the film. It didn't make much money in its day, but it does seem to be well-liked over time."

In December 2022, journalist Arthur Cios tweeted that, during an interview for Avatar: The Way of Water, he asked Cameron about a 4K release of The Abyss and that Cameron had told him "he had a new master and it would be out by March 2023 max." However, Cameron conceded in January 2023 that a 4K release would be pushed back to the "second half of 2023" alongside True Lies. Cameron premiered the completed 4K remaster at Beyond Fest in September 2023 and said new home video releases were coming in "a couple of months". Walt Disney Studios Home Entertainment released the film on Blu-ray and Ultra HD Blu-ray on March 12, 2024. Along with including both cuts, the set has several new special features and includes all of the special features from the previous 2000 DVD release. A United Kingdom release for the 4K restoration was cancelled due to Cameron's refusal to cut the rat breathing liquid scene.

Adaptations
American science fiction author Orson Scott Card was hired to write a novelization of the film based on the screenplay and discussions with Cameron. He wrote back-stories for Bud, Lindsey, and Coffey as a means not only of helping the actors define their roles, but also to justify some of their behavior and mannerisms in the film. Card also wrote the aliens as a colonizing species which preferentially sought high-pressure deep-water worlds to build their ships as they traveled further into the galaxy (their mothership was in orbit on the far side of the Moon). The NTIs' knowledge of neuroanatomy and nanoscale manipulation of biochemistry was responsible for many aspects of the film.

A licensed interactive fiction video game based on the script was being developed for Infocom by Bob Bates, but was cancelled when Infocom was shut down by its then-parent company Activision. Sound Source Interactive later released an action video game in 1998 entitled The Abyss: Incident at Europa. The game takes place a few years after the film, where the player must find a cure for a deadly virus.

A two-issue comic book adaptation was published by Dark Horse Comics.

title: The Abyss


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Dedekind cut

In mathematics, Dedekind cuts, named after German mathematician Richard Dedekind (but previously considered by Joseph Bertrand ), are а method of construction of the real numbers from the rational numbers. A Dedekind cut is a partition of the rational numbers into two sets A and B, such that each element of A is less than every element of B, and A contains no greatest element. The set B may or may not have a smallest element among the rationals. If B has a smallest element among the rationals, the cut corresponds to that rational. Otherwise, that cut defines a unique irrational number which, loosely speaking, fills the "gap" between A and B. In other words, A contains every rational number less than the cut, and B contains every rational number greater than or equal to the cut. An irrational cut is equated to an irrational number which is in neither set. Every real number, rational or not, is equated to one and only one cut of rationals.

Dedekind cuts can be generalized from the rational numbers to any totally ordered set by defining a Dedekind cut as a partition of a totally ordered set into two non-empty parts A and B, such that A is closed downwards (meaning that for all a in A, x ≤ a implies that x is in A as well) and B is closed upwards, and A contains no greatest element. See also completeness (order theory).

It is straightforward to show that a Dedekind cut among the real numbers is uniquely defined by the corresponding cut among the rational numbers. Similarly, every cut of reals is identical to the cut produced by a specific real number (which can be identified as the smallest element of the B set). In other words, the number line where every real number is defined as a Dedekind cut of rationals is a complete continuum without any further gaps.

Definition
A Dedekind cut is a partition of the rationals $$\mathbb{Q}$$ into two subsets $$A$$ and $$B$$ such that

 * 1) $$A$$ is nonempty.
 * 2) $$A \neq \mathbb{Q}$$ (equivalently, $$B$$ is nonempty).
 * 3) If $$x, y \in \mathbb{Q}$$, $$ x < y $$, and $$ y \in A $$, then $$ x \in A $$. ($$A$$ is "closed downwards".)
 * 4) If $$ x \in A $$, then there exists a $$ y \in A $$ such that $$ y > x $$. ($$A$$ does not contain a greatest element.)

By omitting the first two requirements, we formally obtain the extended real number line.

Representations
It is more symmetrical to use the (A, B) notation for Dedekind cuts, but each of A and B does determine the other. It can be a simplification, in terms of notation if nothing more, to concentrate on one "half" — say, the lower one — and call any downward-closed set A without greatest element a "Dedekind cut".

If the ordered set S is complete, then, for every Dedekind cut (A, B) of S, the set B must have a minimal element b, hence we must have that A is the interval (−∞, b), and B the interval [b, +∞). In this case, we say that b is represented by the cut (A, B).

The important purpose of the Dedekind cut is to work with number sets that are not complete. The cut itself can represent a number not in the original collection of numbers (most often rational numbers). The cut can represent a number b, even though the numbers contained in the two sets A and B do not actually include the number b that their cut represents.

For example if A and B only contain rational numbers, they can still be cut at $$\sqrt{2}$$ by putting every negative rational number in A, along with every non-negative rational number whose square is less than 2; similarly B would contain every positive rational number whose square is greater than or equal to 2. Even though there is no rational value for $$\sqrt{2}$$, if the rational numbers are partitioned into A and B this way, the partition itself represents an irrational number.

Ordering of cuts
Regard one Dedekind cut (A, B) as less than another Dedekind cut (C, D) (of the same superset) if A is a proper subset of C. Equivalently, if D is a proper subset of B, the cut (A, B) is again less than (C, D). In this way, set inclusion can be used to represent the ordering of numbers, and all other relations (greater than, less than or equal to, equal to, and so on) can be similarly created from set relations.

The set of all Dedekind cuts is itself a linearly ordered set (of sets). Moreover, the set of Dedekind cuts has the least-upper-bound property, i.e., every nonempty subset of it that has any upper bound has a least upper bound. Thus, constructing the set of Dedekind cuts serves the purpose of embedding the original ordered set S, which might not have had the least-upper-bound property, within a (usually larger) linearly ordered set that does have this useful property.

Construction of the real numbers
A typical Dedekind cut of the rational numbers $$\Q$$ is given by the partition $$(A,B)$$ with

 * $$A = \{ a\in\mathbb{Q} : a^2 < 2 \text{ or } a < 0 \},$$
 * $$B = \{ b\in\mathbb{Q} : b^2 \ge 2 \text{ and } b \ge 0 \}.$$

This cut represents the irrational number $$\sqrt{2}$$ in Dedekind's construction. The essential idea is that we use a set $$A$$, which is the set of all rational numbers whose squares are less than 2, to "represent" number $$\sqrt{2}$$, and further, by defining properly arithmetic operators over these sets (addition, subtraction, multiplication, and division), these sets (together with these arithmetic operations) form the familiar real numbers.

To establish this, one must show that $$A$$ really is a cut (according to the definition) and the square of $$A$$, that is $$A \times A$$ (please refer to the link above for the precise definition of how the multiplication of cuts is defined), is $$2$$ (note that rigorously speaking this number 2 is represented by a cut $$\{x\ |\ x \in \mathbb{Q}, x < 2\}$$). To show the first part, we show that for any positive rational $$x$$ with $$x^2 < 2$$, there is a rational $$y$$ with $$x < y$$ and $$y^2 < 2$$. The choice $$y=\frac{2x+2}{x+2}$$ works, thus $$A$$ is indeed a cut. Now armed with the multiplication between cuts, it is easy to check that $$A \times A \le 2$$ (essentially, this is because $$x \times y \le 2, \forall x, y \in A, x, y \ge 0$$). Therefore to show that $$A \times A = 2$$, we show that $$A \times A \ge 2$$, and it suffices to show that for any $$r < 2$$, there exists $$x \in A$$, $$x^2 > r$$. For this we notice that if $$x > 0, 2-x^2=\epsilon > 0$$, then $$2-y^2 \le \frac{\epsilon}{2}$$ for the $$y$$ constructed above, this means that we have a sequence in $$A$$ whose square can become arbitrarily close to $$2$$, which finishes the proof.

Note that the equality $b^{2} = 2$ cannot hold since $\sqrt{2}$ is not rational.

Relation to interval arithmetic
Given a Dedekind cut representing the real number $$r$$ by splitting the rationals into $$(A,B)$$ where rationals in $$A$$ are less than $$r$$ and rationals in $$B$$ are greater than $$r$$, it can be equivalently represented as the set of pairs $$(a,b)$$ with $$a \in A$$ and $$b \in B$$, with the lower cut and the upper cut being given by projections. This corresponds exactly to the set of intervals approximating $$r$$.

This allows the basic arithmetic operations on the real numbers to be defined in terms of interval arithmetic. This property and its relation with real numbers given only in terms of $$A$$ and $$B$$ is particularly important in weaker foundations such as constructive analysis.

Arbitrary linearly ordered sets
In the general case of an arbitrary linearly ordered set X, a cut is a pair $$(A,B)$$ such that $$A \cup B = X $$ and $$a \in A$$, $$b \in B$$ imply $$a < b$$. Some authors add the requirement that both A and B are nonempty.

If neither A has a maximum, nor B has a minimum, the cut is called a gap. A linearly ordered set endowed with the order topology is compact if and only if it has no gap.

Surreal numbers
A construction resembling Dedekind cuts is used for (one among many possible) constructions of surreal numbers. The relevant notion in this case is a Cuesta-Dutari cut, named after the Spanish mathematician Norberto Cuesta Dutari.

Partially ordered sets
More generally, if S is a partially ordered set, a completion of S means a complete lattice L with an order-embedding of S into L. The notion of complete lattice generalizes the least-upper-bound property of the reals.

One completion of S is the set of its downwardly closed subsets, ordered by inclusion. A related completion that preserves all existing sups and infs of S is obtained by the following construction: For each subset A of S, let Au denote the set of upper bounds of A, and let Al denote the set of lower bounds of A. (These operators form a Galois connection.) Then the Dedekind–MacNeille completion of S consists of all subsets A for which (Au)l = A; it is ordered by inclusion. The Dedekind-MacNeille completion is the smallest complete lattice with S embedded in it.

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/

title: Dedekind cut


text:
DNA vaccine

A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.

DNA vaccines work by injecting genetically engineered plasmid containing the DNA sequence encoding the antigen(s) against which an immune response is sought, so the cells directly produce the antigen, thus causing a protective immunological response. DNA vaccines have theoretical advantages over conventional vaccines, including the "ability to induce a wider range of types of immune response". Several DNA vaccines have been tested for veterinary use. In some cases, protection from disease in animals has been obtained, in others not. Research is ongoing over the approach for viral, bacterial and parasitic diseases in humans, as well as for cancers. In August 2021, Indian authorities gave emergency approval to ZyCoV-D. Developed by Cadila Healthcare, it is the first DNA vaccine approved for humans.

History
Conventional vaccines contain either specific antigens from a pathogen, or attenuated viruses which stimulate an immune response in the vaccinated organism. DNA vaccines are members of the genetic vaccines, because they contain a genetic information (DNA or RNA) that codes for the cellular production (protein biosynthesis) of an antigen. DNA vaccines contain DNA that codes for specific antigens from a pathogen. The DNA is injected into the body and taken up by cells, whose normal metabolic processes synthesize proteins based on the genetic code in the plasmid that they have taken up. Because these proteins contain regions of amino acid sequences that are characteristic of bacteria or viruses, they are recognized as foreign and when they are processed by the host cells and displayed on their surface, the immune system is alerted, which then triggers immune responses. Alternatively, the DNA may be encapsulated in protein to facilitate cell entry. If this capsid protein is included in the DNA, the resulting vaccine can combine the potency of a live vaccine without reversion risks.

In 1983, Enzo Paoletti and Dennis Panicali at the New York Department of Health devised a strategy to produce recombinant DNA vaccines by using genetic engineering to transform ordinary smallpox vaccine into vaccines that may be able to prevent other diseases. They altered the DNA of cowpox virus by inserting a gene from other viruses (namely Herpes simplex virus, hepatitis B and influenza). In 1993, Jeffrey Ulmer and co-workers at Merck Research Laboratories demonstrated that direct injection of mice with plasmid DNA encoding a flu antigen protected the animals against subsequent experimental infection with influenza virus. In 2016 a DNA vaccine for the Zika virus began testing in humans at the National Institutes of Health. The study was planned to involve up to 120 subjects aged between 18 and 35. Separately, Inovio Pharmaceuticals and GeneOne Life Science began tests of a different DNA vaccine against Zika in Miami. The NIH vaccine is injected into the upper arm under high pressure. Manufacturing the vaccines in volume remained unsolved as of August 2016. Clinical trials for DNA vaccines to prevent HIV are underway.

In August 2021, Indian authorities gave emergency approval to ZyCoV-D. Developed by Cadila Healthcare, it is the first DNA vaccine against COVID-19.

Applications
no DNA vaccines have been approved for human use in the United States. Few experimental trials have evoked a response strong enough to protect against disease and the technique's usefulness remains to be proven in humans.

A veterinary DNA vaccine to protect horses from West Nile virus has been approved. Another West Nile virus vaccine has been tested successfully on American robins.

DNA immunization is also being investigated as a means of developing antivenom sera. DNA immunization can be used as a technology platform for monoclonal antibody induction.

Advantages

 * No risk for infections
 * Antigen presentation by both MHC class I and class II molecules
 * Polarise T-cell response toward type 1 or type 2
 * Immune response focused on the antigen of interest
 * Ease of development and production
 * Stability for storage and shipping
 * Cost-effectiveness
 * Obviates need for peptide synthesis, expression and purification of recombinant proteins and use of toxic adjuvants
 * Long-term persistence of immunogen
 * In vivo expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications

Disadvantages

 * Limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides)
 * Potential for atypical processing of bacterial and parasite proteins
 * Potential when using nasal spray administration of plasmid DNA nanoparticles to transfect non-target cells, such as brain cells
 * Cross-contamination when manufacturing different types of live vaccines in same facility

Vector design
DNA vaccines elicit the best immune response when high-expression vectors are used. These are plasmids that usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest. Intron A may sometimes be included to improve mRNA stability and hence increase protein expression. Plasmids also include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences. Polycistronic vectors (with multiple genes of interest) are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.

Because the plasmid – carrying relatively small genetic code up to about 200 Kbp – is the "vehicle" from which the immunogen is expressed, optimising vector design for maximal protein expression is essential. One way of enhancing protein expression is by optimising the codon usage of pathogenic mRNAs for eukaryotic cells. Pathogens often have different AT-contents than the target species, so altering the gene sequence of the immunogen to reflect the codons more commonly used in the target species may improve its expression.

Another consideration is the choice of promoter. The SV40 promoter was conventionally used until research showed that vectors driven by the Rous Sarcoma Virus (RSV) promoter had much higher expression rates. More recently, expression and immunogenicity have been further increased in model systems by the use of the cytomegalovirus (CMV) immediate early promoter, and a retroviral cis-acting transcriptional element. Additional modifications to improve expression rates include the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and modifications to the polyadenylation and transcriptional termination sequences. An example of DNA vaccine plasmid is pVAC, which uses SV40 promoter.

Structural instability phenomena are of particular concern for plasmid manufacture, DNA vaccination and gene therapy. Accessory regions pertaining to the plasmid backbone may engage in a wide range of structural instability phenomena. Well-known catalysts of genetic instability include direct, inverted and tandem repeats, which are conspicuous in many commercially available cloning and expression vectors. Therefore, the reduction or complete elimination of extraneous noncoding backbone sequences would pointedly reduce the propensity for such events to take place and consequently the overall plasmid's recombinogenic potential.

Mechanism of plasmids
Once the plasmid inserts itself into the transfected cell nucleus, it codes for a peptide string of a foreign antigen. On its surface the cell displays the foreign antigen with both histocompatibility complex (MHC) classes I and class II molecules. The antigen-presenting cell then travels to the lymph nodes and presents the antigen peptide and costimulatory molecule signalling to T-cell, initiating the immune response.

Vaccine insert design
Immunogens can be targeted to various cellular compartments to improve antibody or cytotoxic T-cell responses. Secreted or plasma membrane-bound antigens are more effective at inducing antibody responses than cytosolic antigens, while cytotoxic T-cell responses can be improved by targeting antigens for cytoplasmic degradation and subsequent entry into the major histocompatibility complex (MHC) class I pathway. This is usually accomplished by the addition of N-terminal ubiquitin signals.

The conformation of the protein can also affect antibody responses. "Ordered" structures (such as viral particles) are more effective than unordered structures. Strings of minigenes (or MHC class I epitopes) from different pathogens raise cytotoxic T-cell responses to some pathogens, especially if a TH epitope is also included.

Delivery
DNA vaccines have been introduced into animal tissues by multiple methods. In 1999, the two most popular approaches were injection of DNA in saline: by using a standard hypodermic needle, or by using a gene gun delivery. Several other techniques have been documented in the intervening years.

Saline injection
Injection in saline is normally conducted intramuscularly (IM) in skeletal muscle, or intradermally (ID), delivering DNA to extracellular spaces. This can be assisted either 1) by electroporation; 2) by temporarily damaging muscle fibres with myotoxins such as bupivacaine; or 3) by using hypertonic solutions of saline or sucrose. Immune responses to this method can be affected by factors including needle type, needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the recipient.

Gene gun
Gene gun delivery ballistically accelerates plasmid DNA (pDNA) that has been absorbed onto gold or tungsten microparticles into the target cells, using compressed helium as an accelerant.

Mucosal surface delivery
Alternatives included aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa, and topical administration of pDNA to the eye and vaginal mucosa. Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Salmonalla, Shigella or Listeria vectors for oral administration to the intestinal mucosa and recombinant adenovirus vectors.

Polymer vehicle
A hybrid vehicle composed of bacteria cell and synthetic polymers has been employed for DNA vaccine delivery. An E. coli inner core and poly(beta-amino ester) outer coat function synergistically to increase efficiency by addressing barriers associated with antigen-presenting cell gene delivery which include cellular uptake and internalization, phagosomal escape and intracellular cargo concentration. Tested in mice, the hybrid vector was found to induce immune response.

ELI immunization
Another approach to DNA vaccination is expression library immunization (ELI). Using this technique, potentially all the genes from a pathogen can be delivered at one time, which may be useful for pathogens that are difficult to attenuate or culture. ELI can be used to identify which genes induce a protective response. This has been tested with Mycoplasma pulmonis, a murine lung pathogen with a relatively small genome. Even partial expression libraries can induce protection from subsequent challenge.

Dosage
The delivery method determines the dose required to raise an effective immune response. Saline injections require variable amounts of DNA, from 10 μg to 1 mg, whereas gene gun deliveries require 100 to 1000 times less. Generally, 0.2 μg – 20 μg are required, although quantities as low as 16 ng have been reported. These quantities vary by species. Mice for example, require approximately 10 times less DNA than primates. Saline injections require more DNA because the DNA is delivered to the extracellular spaces of the target tissue (normally muscle), where it has to overcome physical barriers (such as the basal lamina and large amounts of connective tissue) before it is taken up by the cells, while gene gun deliveries drive/force DNA directly into the cells, resulting in less "wastage".

Helper T cell responses
DNA immunization can raise multiple TH responses, including lymphoproliferation and the generation of a variety of cytokine profiles. A major advantage of DNA vaccines is the ease with which they can be manipulated to bias the type of T-cell help towards a TH1 or TH2 response. Each type has distinctive patterns of lymphokine and chemokine expression, specific types of immunoglobulins, patterns of lymphocyte trafficking and types of innate immune responses.

Other types of T-cell help
The type of T-cell help raised is influenced by the delivery method and the type of immunogen expressed, as well as the targeting of different lymphoid compartments. Generally, saline needle injections (either IM or ID) tend to induce TH1 responses, while gene gun delivery raises TH2 responses. This is true for intracellular and plasma membrane-bound antigens, but not for secreted antigens, which seem to generate TH2 responses, regardless of the method of delivery.

Generally the type of T-cell help raised is stable over time, and does not change when challenged or after subsequent immunizations that would normally have raised the opposite type of response in a naïve specimen. However, Mor et al.. (1995) immunized and boosted mice with pDNA encoding the circumsporozoite protein of the mouse malarial parasite Plasmodium yoelii (PyCSP) and found that the initial TH2 response changed, after boosting, to a TH1 response.

Basis for different types of T-cell help
How these different methods operate, the forms of antigen expressed, and the different profiles of T-cell help is not understood. It was thought that the relatively large amounts of DNA used in IM injection were responsible for the induction of TH1 responses. However, evidence shows no dose-related differences in TH type. The type of T-cell help raised is determined by the differentiated state of antigen presenting cells. Dendritic cells can differentiate to secrete IL-12 (which supports TH1 cell development) or IL-4 (which supports TH2 responses). pDNA injected by needle is endocytosed into the dendritic cell, which is then stimulated to differentiate for TH1 cytokine (IL-12) production, while the gene gun bombards the DNA directly into the cell, thus bypassing TH1 stimulation.

Practical uses of polarised T-cell help
Polarisation in T-cell help is useful in influencing allergic responses and autoimmune diseases. In autoimmune diseases, the goal is to shift the self-destructive TH1 response (with its associated cytotoxic T cell activity) to a non-destructive TH2 response. This has been successfully applied in predisease priming for the desired type of response in preclinical models and is somewhat successful in shifting the response for an established disease.

Cytotoxic T-cell responses
One of the advantages of DNA vaccines is that they are able to induce cytotoxic T lymphocytes (CTL) without the inherent risk associated with live vaccines. CTL responses can be raised against immunodominant and immunorecessive CTL epitopes, as well as subdominant CTL epitopes, in a manner that appears to mimic natural infection. This may prove to be a useful tool in assessing CTL epitopes and their role in providing immunity.

Cytotoxic T-cells recognise small peptides (8-10 amino acids) complexed to MHC class I molecules. These peptides are derived from cytosolic proteins that are degraded and delivered to the nascent MHC class I molecule within the endoplasmic reticulum (ER). Targeting gene products directly to the ER (by the addition of an ER insertion signal sequence at the N-terminus) should thus enhance CTL responses. This was successfully demonstrated using recombinant vaccinia viruses expressing influenza proteins, but the principle should also be applicable to DNA vaccines. Targeting antigens for intracellular degradation (and thus entry into the MHC class I pathway) by the addition of ubiquitin signal sequences, or mutation of other signal sequences, was shown to be effective at increasing CTL responses.

CTL responses can be enhanced by co-inoculation with co-stimulatory molecules such as B7-1 or B7-2 for DNA vaccines against influenza nucleoprotein, or GM-CSF for DNA vaccines against the murine malaria model P. yoelii. Co-inoculation with plasmids encoding co-stimulatory molecules IL-12 and TCA3 were shown to increase CTL activity against HIV-1 and influenza nucleoprotein antigens.

Humoral (antibody) response
Antibody responses elicited by DNA vaccinations are influenced by multiple variables, including antigen type; antigen location (i.e. intracellular vs. secreted); number, frequency and immunization dose; site and method of antigen delivery.

Kinetics of antibody response
Humoral responses after a single DNA injection can be much longer-lived than after a single injection with a recombinant protein. Antibody responses against hepatitis B virus (HBV) envelope protein (HBsAg) have been sustained for up to 74 weeks without boost, while lifelong maintenance of protective response to influenza haemagglutinin was demonstrated in mice after gene gun delivery. Antibody-secreting cells (ASC) migrate to the bone marrow and spleen for long-term antibody production, and generally localise there after one year.

Comparisons of antibody responses generated by natural (viral) infection, immunization with recombinant protein and immunization with pDNA are summarised in Table 4. DNA-raised antibody responses rise much more slowly than when natural infection or recombinant protein immunization occurs. As many as 12 weeks may be required to reach peak titres in mice, although boosting can decrease the interval. This response is probably due to the low levels of antigen expressed over several weeks, which supports both DNA vaccine expressing HBV small and middle envelope protein was injected into adults with chronic hepatitis. The vaccine resulted in specific interferon gamma cell production. Also specific T-cells for middle envelop proteins antigens were developed. The immune response of the patients was not robust enough to control HBV infection

Additionally, the titres of specific antibodies raised by DNA vaccination are lower than those obtained after vaccination with a recombinant protein. However, DNA immunization-induced antibodies show greater affinity to native epitopes than recombinant protein-induced antibodies. In other words, DNA immunization induces a qualitatively superior response. Antibodies can be induced after one vaccination with DNA, whereas recombinant protein vaccinations generally require a boost. DNA immunization can be used to bias the TH profile of the immune response and thus the antibody isotype, which is not possible with either natural infection or recombinant protein immunization. Antibody responses generated by DNA are useful as a preparative tool. For example, polyclonal and monoclonal antibodies can be generated for use as reagents.

DNA uptake mechanism
When DNA uptake and subsequent expression was first demonstrated in vivo in muscle cells, these cells were thought to be unique because of their extensive network of T-tubules. Using electron microscopy, it was proposed that DNA uptake was facilitated by caveolae (or, non-clathrin coated pits). However, subsequent research revealed that other cells (such as keratinocytes, fibroblasts and epithelial Langerhans cells) could also internalize DNA. The mechanism of DNA uptake is not known.

Two theories dominate – that in vivo uptake of DNA occurs non-specifically, in a method similar to phago- or pinocytosis, or through specific receptors. These might include a 30kDa surface receptor, or The 30kDa surface receptor binds specifically to 4500-bp DNA fragments (which are then internalised) and is found on professional APCs and T-cells. Macrophage scavenger receptors bind to a variety of macromolecules, including polyribonucleotides and are thus candidates for DNA uptake. Receptor-mediated DNA uptake could be facilitated by Gene gun delivery systems, cationic liposome packaging, and other delivery methods bypass this entry method, but understanding it may be useful in reducing costs (e.g. by reducing the requirement for cytofectins), which could be important in animal husbandry.

Antigen presentation by bone marrow-derived cells

Studies using chimeric mice have shown that antigen is presented by bone-marrow derived cells, which include dendritic cells, macrophages and specialised B-cells called professional antigen presenting cells (APC). After gene gun inoculation to the skin, transfected Langerhans cells migrate to the draining lymph node to present antigens. After IM and ID injections, dendritic cells present antigen in the draining lymph node and transfected macrophages have been found in the peripheral blood.

Besides direct transfection of dendritic cells or macrophages, cross priming occurs following IM, ID and gene gun DNA deliveries. Cross-priming occurs when a bone marrow-derived cell presents peptides from proteins synthesised in another cell in the context of MHC class 1. This can prime cytotoxic T-cell responses and seems to be important for a full primary immune response.

Target site role
IM and ID DNA delivery initiate immune responses differently. In the skin, keratinocytes, fibroblasts and Langerhans cells take up and express antigens and are responsible for inducing a primary antibody response. Transfected Langerhans cells migrate out of the skin (within 12 hours) to the draining lymph node where they prime secondary B- and T-cell responses. In skeletal muscle, striated muscle cells are most frequently transfected, but seem to be unimportant in immune response. Instead, IM inoculated DNA "washes" into the draining lymph node within minutes, where distal dendritic cells are transfected and then initiate an immune response. Transfected myocytes seem to act as a "reservoir" of antigen for trafficking professional APCs.

Maintenance of immune response
DNA vaccination generates an effective immune memory via the display of antigen-antibody complexes on follicular dendritic cells (FDC), which are potent B-cell stimulators. T-cells can be stimulated by similar, germinal centre dendritic cells. FDC are able to generate an immune memory because antibodies production "overlaps" long-term expression of antigen, allowing antigen-antibody immunocomplexes to form and be displayed by FDC.

Interferons
Both helper and cytotoxic T-cells can control viral infections by secreting interferons. Cytotoxic T cells usually kill virally infected cells. However, they can also be stimulated to secrete antiviral cytokines such as IFN-γ and TNF-α, which do not kill the cell, but limit viral infection by down-regulating the expression of viral components. DNA vaccinations can be used to curb viral infections by non-destructive IFN-mediated control. This was demonstrated for hepatitis B. IFN-γ is critically important in controlling malaria infections and is a consideration for anti-malarial DNA vaccines.

Cytokine modulation
An effective vaccine must induce an appropriate immune response for a given pathogen. DNA vaccines can polarise T-cell help towards TH1 or TH2 profiles and generate CTL and/or antibody when required. This can be accomplished by modifications to the form of antigen expressed (i.e. intracellular vs. secreted), the method and route of delivery or the dose. It can also be accomplished by the co-administration of plasmid DNA encoding immune regulatory molecules, i.e. cytokines, lymphokines or co-stimulatory molecules. These "genetic adjuvants" can be administered as a: In general, co-administration of pro-inflammatory agents (such as various interleukins, tumor necrosis factor, and GM-CSF) plus TH2-inducing cytokines increase antibody responses, whereas pro-inflammatory agents and TH1-inducing cytokines decrease humoral responses and increase cytotoxic responses (more important in viral protection). Co-stimulatory molecules such as B7-1, B7-2 and CD40L are sometimes used.
 * mixture of 2 plasmids, one encoding the immunogen and the other encoding the cytokine
 * single bi- or polycistronic vector, separated by spacer regions
 * plasmid-encoded chimera, or fusion protein

This concept was applied in topical administration of pDNA encoding IL-10. Plasmid encoding B7-1 (a ligand on APCs) successfully enhanced the immune response in tumour models. Mixing plasmids encoding GM-CSF and the circumsporozoite protein of P. yoelii (PyCSP) enhanced protection against subsequent challenge (whereas plasmid-encoded PyCSP alone did not). It was proposed that GM-CSF caused dendritic cells to present antigen more efficiently and enhance IL-2 production and TH cell activation, thus driving the increased immune response. This can be further enhanced by first priming with a pPyCSP and pGM-CSF mixture, followed by boosting with a recombinant poxvirus expressing PyCSP. However, co-injection of plasmids encoding GM-CSF (or IFN-γ, or IL-2) and a fusion protein of P. chabaudi merozoite surface protein 1 (C-terminus)-hepatitis B virus surface protein (PcMSP1-HBs) abolished protection against challenge, compared to protection acquired by delivery of pPcMSP1-HBs alone.

The advantages of genetic adjuvants are their low cost and simple administration, as well as avoidance of unstable recombinant cytokines and potentially toxic, "conventional" adjuvants (such as alum, calcium phosphate, monophosphoryl lipid A, cholera toxin, cationic and mannan-coated liposomes, QS21, carboxymethyl cellulose and ubenimix). However, the potential toxicity of prolonged cytokine expression is not established. In many commercially important animal species, cytokine genes have not been identified and isolated. In addition, various plasmid-encoded cytokines modulate the immune system differently according to the delivery time. For example, some cytokine plasmid DNAs are best delivered after immunogen pDNA, because pre- or co-delivery can decrease specific responses and increase non-specific responses.

Immunostimulatory CpG motifs
Plasmid DNA itself appears to have an adjuvant effect on the immune system. Bacterially derived DNA can trigger innate immune defence mechanisms, the activation of dendritic cells and the production of TH1 cytokines. This is due to recognition of certain CpG dinucleotide sequences that are immunostimulatory. CpG stimulatory (CpG-S) sequences occur twenty times more frequently in bacterially-derived DNA than in eukaryotes. This is because eukaryotes exhibit "CpG suppression" – i.e. CpG dinucleotide pairs occur much less frequently than expected. Additionally, CpG-S sequences are hypomethylated. This occurs frequently in bacterial DNA, while CpG motifs occurring in eukaryotes are methylated at the cytosine nucleotide. In contrast, nucleotide sequences that inhibit the activation of an immune response (termed CpG neutralising, or CpG-N) are over represented in eukaryotic genomes. The optimal immunostimulatory sequence is an unmethylated CpG dinucleotide flanked by two 5’ purines and two 3’ pyrimidines. Additionally, flanking regions outside this immunostimulatory hexamer must be guanine-rich to ensure binding and uptake into target cells.

The innate system works with the adaptive immune system to mount a response against the DNA encoded protein. CpG-S sequences induce polyclonal B-cell activation and the upregulation of cytokine expression and secretion. Stimulated macrophages secrete IL-12, IL-18, TNF-α, IFN-α, IFN-β and IFN-γ, while stimulated B-cells secrete IL-6 and some IL-12.

Manipulation of CpG-S and CpG-N sequences in the plasmid backbone of DNA vaccines can ensure the success of the immune response to the encoded antigen and drive the immune response toward a TH1 phenotype. This is useful if a pathogen requires a TH response for protection. CpG-S sequences have also been used as external adjuvants for both DNA and recombinant protein vaccination with variable success rates. Other organisms with hypomethylated CpG motifs have demonstrated the stimulation of polyclonal B-cell expansion. The mechanism behind this may be more complicated than simple methylation – hypomethylated murine DNA has not been found to mount an immune response.

Most of the evidence for immunostimulatory CpG sequences comes from murine studies. Extrapolation of this data to other species requires caution – individual species may require different flanking sequences, as binding specificities of scavenger receptors vary across species. Additionally, species such as ruminants may be insensitive to immunostimulatory sequences due to their large gastrointestinal load.

Alternative boosts
DNA-primed immune responses can be boosted by the administration of recombinant protein or recombinant poxviruses. "Prime-boost" strategies with recombinant protein have successfully increased both neutralising antibody titre, and antibody avidity and persistence, for weak immunogens, such as HIV-1 envelope protein. Recombinant virus boosts have been shown to be very efficient at boosting DNA-primed CTL responses. Priming with DNA focuses the immune response on the required immunogen, while boosting with the recombinant virus provides a larger amount of expressed antigen, leading to a large increase in specific CTL responses.

Prime-boost strategies have been successful in inducing protection against malarial challenge in a number of studies. Primed mice with plasmid DNA encoding Plasmodium yoelii circumsporozoite surface protein (PyCSP), then boosted with a recombinant vaccinia virus expressing the same protein had significantly higher levels of antibody, CTL activity and IFN-γ, and hence higher levels of protection, than mice immunized and boosted with plasmid DNA alone. This can be further enhanced by priming with a mixture of plasmids encoding PyCSP and murine GM-CSF, before boosting with recombinant vaccinia virus. An effective prime-boost strategy for the simian malarial model P. knowlesi has also been demonstrated. Rhesus monkeys were primed with a multicomponent, multistage DNA vaccine encoding two liver-stage antigens – the circumsporozoite surface protein (PkCSP) and sporozoite surface protein 2 (PkSSP2) – and two blood stage antigens – the apical merozoite surface protein 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42). They were then boosted with a recombinant canarypox virus encoding all four antigens (ALVAC-4). Immunized monkeys developed antibodies against sporozoites and infected erythrocytes, and IFN-γ-secreting T-cell responses against peptides from PkCSP. Partial protection against sporozoite challenge was achieved, and mean parasitemia was significantly reduced, compared to control monkeys. These models, while not ideal for extrapolation to P. falciparum in humans, will be important in pre-clinical trials.

DNA
The efficiency of DNA immunization can be improved by stabilising DNA against degradation, and increasing the efficiency of delivery of DNA into antigen-presenting cells. This has been demonstrated by coating biodegradable cationic microparticles (such as poly(lactide-co-glycolide) formulated with cetyltrimethylammonium bromide) with DNA. Such DNA-coated microparticles can be as effective at raising CTL as recombinant viruses, especially when mixed with alum. Particles 300 nm in diameter appear to be most efficient for uptake by antigen presenting cells.

Alphavirus vectors
Recombinant alphavirus-based vectors have been used to improve DNA vaccination efficiency. The gene encoding the antigen of interest is inserted into the alphavirus replicon, replacing structural genes but leaving non-structural replicase genes intact. The Sindbis virus and Semliki Forest virus have been used to build recombinant alphavirus replicons. Unlike conventional DNA vaccinations alphavirus vectors kill transfected cells and are only transiently expressed. Alphavirus replicase genes are expressed in addition to the vaccine insert. It is not clear how alphavirus replicons raise an immune response, but it may be due to the high levels of protein expressed by this vector, replicon-induced cytokine responses, or replicon-induced apoptosis leading to enhanced antigen uptake by dendritic cells.

title: DNA vaccine


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 * Planung - European Journal of Planning
 * Cyburbia - The planning portal
 * I parchi italiani

 * WebStrade E-magazine
 * US Department of Transportation
 * Transportation Reasearch Board
 * Turner-Fairbank Highway Research Center (TFHRC)
 * Modern Roundabouts
 * FHWA - Roundabouts: An Informational Guide (pdf)
 * Manual of Traffic Signs
 * Manual on Uniform Traffic Control Devices (MUTCD)
 * MUTCD - Standard Highway Signs (pdf)
 * The UK highway code
 * Linee TAV

 * Fondazione Ricovero Martinelli Onlus - Cinisello Balsamo

title: User:Dch