By David N. Leff
The dog may be man's best friend, but in most of the world dogs are also his worst (nonhuman) enemy.
In developing countries, where dogs run loose, more than 40,000 people a year die of bites from rabid canines. In the U.S., where domestic animals are vaccinated against rabies, this viral infection has killed only eight people since 1994.
Two of them were in South Texas, along the Mexican border, where rabid wild coyotes bite tame dogs, which often attack people. So do rabies-infected raccoons, skunks, foxes and bats. Victims thus exposed have a better chance of surviving when vaccinated in time with a human diploid cell vaccine (HDCV), made by attenuating the virulence of rabies virus by culturing in human cells.
Although highly effective, HDCV immunization is expensive (at $2,000 per treatment), and the vaccine has to be kept refrigerated. The cost and handling requirements rule it out in the countries that need it most.
Late last year, the Centers for Disease Control (CDC), in Atlanta, injected a dozen cynomolgous monkeys with full-strength, lethal, wild-type South Texas rabies virus from the salivary gland of a rabid dog. The monkeys had come by air freight from the Rocky Mountain Laboratories of the National Institute of Allergy and Infectious Diseases, in Hamilton, Mont.
There, these animals had taken part in a preclinical trial of the first DNA-based rabies vaccine ever tested in nonhuman primates.
Of the 12-monkey cohort, eight had received the experimental DNA vaccine, two HDCV, and the remaining couple of control animals, non-rabies DNA shots. Ten days later, those last two were showing the early central-nervous-system signs of rabies: lethargy, food refusal, altered vocal sounds, paralysis. To spare them the agonizing endpoints of terminal rabies, the CDC virologists put them to death.
All of the other ten, meanwhile, survived nicely on their DNA or HDCV vaccinations, followed up by booster shots. As the just-published August issue of Nature Medicine reported, "DNA immunization protects nonhuman primates against rabies virus."
Virologist Donald Lodmell, at the Rocky Mountain lab, is the paper's senior author. He told BioWorld Today the DNA vaccine construct centers on a viral gene "that's responsible for the primary immunity to rabies virus. It encodes the protein constituting the glycoprotein spikes that stud the surface of the bullet-shaped virus."
Lodmell and his co-authors inserted this recombinant gene sequence in a plasmid with a cytomegalovirus sequence as promoter.
Antibody, Yes; T Cells, No
"All the neutralizing antibody is made by the immune system against this glycoprotein," Lodmell pointed out. "It's the primary player in immunity to rabies virus. Cytotoxic T lymphocytes have minimal, or no, role in protection.
"It worked really well in mice," he recalled, "so we decided, 'Let's go for the big ones — the monkeys.'"
At the outset, they inoculated four of the 12 animals by needle injection intradermally (through the skin), and four biolistically, by gene gun. Two other subjects received the standard HDCV shots, to compare efficacy with the DNA vaccine. The two controls got only empty plasmids — without the viral gene sequence.
Thirty days later, the HDCV pair had peak levels of neutralizing antibody, whereas the four gene-gun recipients' levels were much lower. But thereafter, the HDCV cohort's levels started going downhill, while the DNA gene-gun contingent's antibody count went up approximately fourfold by day 60.
Meanwhile, the quartet of monkeys that received their DNA vaccine via needle showed no signs of acquiring antibody immunity.
At 190 days into the trial, all animals got booster shots. These brought the HDCV contingent's antibodies up 18-fold, while those of the gene-gun contingent jumped 176-fold. From these record scores, both of those memory-antibody levels gradually declined and equalized over the next 220 days.
As expected, the lethal challenge with virulent live virus, administered at the CDC, drove up the neutralizing-antibody responses of all 10 vaccinated monkeys considerably. Perhaps unexpectedly, six of the eight DNA vaccinees forged ahead of the standard HDCV scores.
Six months after that challenge, all 10 vaccinated primates were killed, and their brains examined for rabies virus antigen. All 10 were negative.
Which led Lodmell to recall "an old saying that DNA immunization was a wonderful vaccination for mice, but it wasn't really working well in subhuman primates. We have evidence to the contrary of that now," he observed.
DNA Vaccine Fits All Global Virus Variants
In a separate survey, the DNA vaccine neutralized 100 percent of 18 rabies virus strains from all over the world. These data, the paper observed, "indicate that a DNA vaccine encoding for the glycoprotein of challenge virus standard could protect against a global spectrum of rabies viruses."
Virologist Charles Rupprecht is chief of the rabies section at the CDC and final author of the Nature Medicine paper. "Part of the problem in the developing countries," he told BioWorld Today, "is the cost of current biologics, and having to maintain their potency under conditions of the cold chain. That would be one of the utilities of these DNA vaccines. Not only low cost, but also thermostability for parenteral injection.
"Another of its utilities," he went on, "would be either for pre-exposure vaccination of people at risk, or for those who have had prior post-exposure vaccination, and now just needed a booster. Or summarily, even the possibilities of transition to dogs, which of course are the rabies reservoirs in most of the developing world. This would be similar to how all rabies vaccines are licensed to animals here in the U.S. Preliminarily, we have started some work in dogs to that effect."
The only drawback of the DNA vaccine," Lodmell allowed, "is that the antibody response cannot be detected before 30 days, which means it wouldn't be useful immediately after exposure. We're working on it," he added.
So are other centers, including the Pasteur Institute, in Paris, where Louis Pasteur created and successfully administered the first rabies vaccine in 1885.
"Our colleagues in Paris," Rupprecht said, "have an alternative DNA construct that they say is similar to this, but — preliminariliy at least in a mouse model — looks as if it may be useful for post-exposure.
"The situation," he went on, "may be dependant on which particular gene or genes are expressed, rather than just the glycoproteins. One may be able to manipulate either by the inclusion of other genes, such as a nuclear protein gene in tandem with the surface one, or perhaps simultaneously from some cytokine genes that might actually enhance virulence of immune effectors.
"So I wouldn't necessarily say on the basis of the pre-exposure work," Rupprecht concluded, "that it has no utility, although it appears it would have less utility based on those preliminary results. We haven't studied this model in a post-exposure series as we have other biologics." *