“And the dogs are exposed how?”
“They have to be inhaling it through those magnificent noses,” I said. “That’s the lowest common denominator of all the cases—they are sniffing around recently-vaccinated cats.”
“And this modified vaccine with the aberrant promoter has no effect on the cat?”
“Not likely,” I aid. “Even if the promoter came from an endogenous cat virus, cats have evolved with the sequence so it is likely harmless, but we actually haven’t had time to look at that.”
Turning back to Anna, Neil asked, “So it’s in the dog, then what?” He had been a Chief Scientific Officer at a major vaccine-producing company. He knew these answers, but he also clearly knew that we would need to be able to explain every detail.
“The promoter triggers the overproduction of GR all over the body, which most immediately sets up an imbalance in the stress feedback loop and sends the dogs into an Addison’s-like adrenal failure.”
Standing again, Neil surveyed Anna’s illustration and pointed to her rendition of a dog. “If the production of GR is in overdrive, wouldn’t everyone researching this be seeing this same increase in the number of mRNA transcripts, or be suspicious of an increase in the GR protein made from them?”
“Sure,” I confirmed.
He eyed me over his shoulder. “So then why hasn’t anyone come to the same conclusion as you, which by the way, I still am not quite clear on.”
“Because it is a normal gene. Everyone is looking for something abnormal,” Anna said.
“But the number of copies would be abnormal,” Neil observed.
“Compared to what?” I asked.
“Compared to what is produced normally,” he said, frustration rising in his voice.
“Right.”
“I’m starting to feel like I’m in an Abbott and Costello routine,” he said as he sat down.
“Everyone and their donkey has been comparing the population of affected individuals with the unaffected,” I said. “But remember, the increase is very slight, and changes in the levels would vary naturally from dog to dog as each responds in a different way to stress.”
“So unless you are comparing the same dog before and after CRFS, which we don’t think anyone is, it wouldn’t be significant,” Anna added.
He looked up at Anna and grinned, and in a congratulatory tone said, “Which you were able to do because you have pre- and post- samples from all the Addison’s dogs who are in your study.”
“Exactly.”
“That still doesn’t explain why no one has at least suspected something from the elevated levels.” He was playing devil’s advocate now.
Anna added her view from the clinical side. “I suspect that when anyone has seen GR elevated they are thinking that this is just how the dogs were reacting to CRFS as their whole system was going out of whack. No one is thinking this is CRFS.”
“Okay, so you have a gene being transcribed at higher than normal rates. How did you connect this back to a contaminated vaccine from a cat?”
“It was surprisingly quick,” I said. “For about a day we thought we hit a brick wall since the gene sequence looked normal. At one point we did start to believe that it was just a symptom, but then Chris had an epiphany, and suggested that we look further upstream and downstream of the GR gene.”
Turning to Chris, Neil said, “And that’s how you connected the dots to the cat endogenous retrovirus?”
“Actually Diana helped. Out of the mouths of babes came the discussion of cross species viruses, evolution, and endogenous retroviruses, so we’re making the kid a co-author.”
“You’re talking to your fourteen-year-old about endogenous retroviruses? Geez, you’re a fun family.” Neil shook his head. “Continue.”
“Actually I wasn’t thinking it was anything as bizarre as what they found. I was simply thinking maybe there’s a yet-to-be-discovered CRFS virus that was influencing transcription of the GR, and that walking up and down the intervening sequences around the GR gene might reveal something.”
I continued, “The GR coding sequence has a typical promoter, 300 bases upstream of the gene, but when we sequenced past that there was another promoter with a small sequence that did not match anything in the dog. Chris and Jamie then had a race to find out what it was, and confirmed within hours that it was from one of the cat’s endogenous retroviruses.”
“That still doesn’t explain how it got into a cat vaccine.”
“It doesn’t. We thought it was from a cat, so we went back to Anna to see if she could scour her records and see if she saw anything going on with cats at the same time. I was hoping that maybe there was something that turned on the endogenous virus in cats, that mildly affected the cat but was deadly to the dog.”
“But that was not the case.” I could tell that he knew.
“Correct,” Anna continued. “When they came to me and told me about the cat sequence, I suspected a vaccine right away because I remembered that several of my cases were very similar. I had a number of clients bring in their dogs dying of CRFS within days of seeing them to vaccinate their cat.” Her shoulders dropped. “I’m still kicking myself for not paying more attention to that.”
Neil gave her a moment before he said, “I suspect if your records had not been so meticulous you wouldn’t have been able to make the connection.” There was a gentleness in his voice that I rarely heard. “So the obvious questions are how did you confirm it and how did it get in the vaccine.” Neil was no longer asking questions. He was walking down the long road of inevitability.
“Let’s start with how it got in the vaccine.” Anna gathered herself and flipped some pages in one of her notebooks. “We think we know that,” she said.
“Of course.” Neil smoothed out Anna’s drawing on the table and placed both his hands on it. He looked Anna squarely in the eyes. “You have the entire thing figured out, don’t you?”
“It sounds masterful, but it is pretty straightforward with all the pieces in front of us.”
“Don’t kid yourselves, millions of dollars are being spent on this, and no one even has a clue, much less a solution.” He sat down next to her and looked around to each of us.
Waving off his declaration, Anna plodded on. “My guess is that a single run or a few lots of the vaccine were contaminated as it was being produced.”
“How? And feel free to detail your lecture for the masses; I think you’re going to be giving a lot of them.”
Inhaling slowly, absorbing the weight of that reality, Anna continued, “This came from the Feline Leukemia Virus or the FeLV vaccine. The vaccine is produced using a modified live virus. They make the vaccine by inoculating cells growing in culture to produce the modified virus and then extract it from the media solution that the cells are growing in.”
“But you think the contaminant is actually a sequence not from FeLV?” Neil asked.
“Uh-huh,” Anna answered. We think that the cell culture that was used to produce the modified FeLV virus was contaminated, but we are only guessing at how. They probably didn’t use a cat cell culture to produce a cat vaccine, but probably something closely related.”
I pulled open a notebook to another set of spreadsheets with numbers and sequences and placed it so both Neil and Anna could see it.
“Contaminated how and with what?” he asked cautiously.
Anna continued. “Well, you know how cells in culture tend to peter out after several generations of replication? They start to accumulate too many mutations and slowly die off?”
“Yes.” Of course he did.
“And you know how there is a technique to sort of immortalize these cultures with endogenous retroviruses from other species which somehow keeps them going.”
“And you think that the cell cultures used to produce the FeLV vaccine became contaminated with a piece of an endogenous retrovirus that was used to immortalize the cell culture.” Neil said.
“Yes. We actually know that the contam
inated vaccine carries the extra promoter sequence. The same extra promoter that appears in the CRFS GR gene.”
Neil stood up and began to pace the living room. He walked across the room and sat down sideways at the piano. Looking down at the keys, he plunked out random notes. We all watched in silence. His expression released and he tilted his head just enough so we could see his face. “I’m taking it that brilliant Anna also keeps vials of all the vaccines she’s used in her clinic.”
She sat up straight and affirmed, “One from every lot.”
“And you, quote, know that the vaccine has an extra promoter because you have already sequenced said vaccine?”
“Every vial,” I confirmed. “Megan is a whiz at the bench and was able to pull the modified FeLV out of each vaccine vial. She found the extra promoter in each one. In a few cases she could confirm from several samples in the same lot.”
“Ah, more than one vial.” Neil locked eyes with Anna. “Nice job.”
“Thank you,” she said without pride.
I motioned him over to come see the printouts. He stood but didn’t move. “You don’t have to look if you don’t want to,” I said. Neil just looked from me to Anna and said nothing. He looked down. Black and white keys: all pretty clear.
I stood quietly left the room and felt Neil move towards her as if swimming upstream against a storm current. Their voices quiet, Anna walked him through the vaccine data. Chris followed me into the kitchen.
His voice hushed, he asked, “You think he is taking this personally?”
“How could he not. He hasn’t asked the question, but he’s brilliant and I’m sure he’s figured it out.”
Returning with a fresh pot of coffee, I put down a trivet, the pot, and a few cups. It was going to be a long night. Chris followed with milk and sugar, and a plate of odd munchies that he pulled from our refrigerator, questionable if edible. Neil stood staring down at Anna’s drawing on the table. We sat down across from him, Anna sat next to him, he remained standing. Anna quietly watched him, her face soft, perhaps with relief because the end was in sight. She caught me staring at her and gave a weak smile.
Neil placed both hands on the table and steeled himself for the question, turning his face slightly and locking eyes with Anna. “The name of the pharmaceutical company on the bottle?”
Anna gently said, “Regnum,” and put her hand on his. He looked crestfallen, even though he had to have known it was coming. This happened under his watch, while he was the Chief Scientific Officer at Regnum. Regardless of whether production of the contaminated vaccine continued after he left, he was in charge when it all started. Quality control and quality assurance fell many rungs down the ladder from where he sat, but he was still ultimately responsible for the quality of the science at Regnum. And he was never one to shy away from responsibility.
“So Regnum caused this, and they hold the key for putting an end to this.”
“They do,” Anna said.
He looked from Anna, to Chris, to me and registered our collective affirmation.
“God damn it,” He said angrily as he pulled his hand away and shut his eyes.
“Yup.” I said. Looking down studying the pattern in the hardwood floors.
We continued on for several more hours. Neil wanted to see everything. Raw data. Photographs of gels showing the increased intensity of mRNA sequences, printouts of sequence data, statistics from sequence comparisons in GenBank. He questioned each conclusion, requiring solid linkage to significant data, without excessive extrapolation. He was relentless in his demand for duplicate results and tight controls and steadfast in his goal to ensure there were no holes in the data and that our conclusions were the only possible outcome. It was clear why he was the CSO at Regnum, past tense noted.
“So, now help me to understand the epidemiology of the outbreak. Why did it start with dog shows?” Neil asked.
Anna showed no signs of becoming exhausted by his challenges, in fact she seemed to be baiting him to keep it going. “Well, it makes sense in terms of concentration of newly vaccinated animals. A lot of cat fanciers keep their cats inside so they don’t fully vaccinate, but once they want to take them to shows, they are required to vaccinate, so with each show there is a significant concentration of the newly vaccinated.”
“How does that relate to dogs that became infected?”
“We looked back at the venues that held dog shows where dogs first came down with CRFS, and most hosted cat shows just prior to the dog shows. In other cases, like where the show was held at 4H centers or county fairs, there are a lot of barn cats, and dog show organizers are very strict about booking only where they guarantee to keep those semi-feral animals vaccinated. There is a lot of concern about rabies and especially parvo as some strains of the virus are believed to have originally come from the cat feline panleukopenia virus. Then there are the shelters. They have a constant stream of cats that are vaccinated upon arrival. A number of shelters have been decimated with cases of CRFS. We haven’t really looked into a lot of cases. They are adding up too fast. But where we have looked, over and over we can make a direct link to recently vaccinated cats. The only thing we haven’t figured out is why it started mainly on the East Coast with a smattering of incidents across the country.”
Chris added, “That’s one of the questions for Regnum, but we suspect that it has to do with the timing of them releasing different vaccine lots and where they were distributed. We are thinking that the contaminated lots were first released regionally to vets, but maybe also to some cat breeders on the East Coast.”
Anna jumped in. “That makes the most sense. Like with show dogs, the cat breeders tend to do their own vaccinations, ordering them online. The question is whether mail order companies also distributed certain lots to certain areas. That would explain there being fewer cases outside the east.”
“For not being epidemiologists, you sure do have a good handle on the spread of the disease,” Neil said, confirming our assumptions. “It is common practice to distribute lots regionally in case of this exact type of situation.” He paused. “It helps to contain any problem that might result in a recall.”
“So Neil, how do you think a vaccine lot got contaminated in the first place?” Chris asked. “We’re not just looking at a common contaminant like a bacterium that might result from unsanitary conditions. It has to be a flaw in the standard practice.”
“I don’t know. You were correct when you said that they probably wouldn’t have used a cat cell line to grow the vaccine. They would have used rabbit kidney cells. And they wouldn’t have used a cat endogenous virus to immortalize the cells. It’s not good practice to use materials from a species to produce a vaccine for that species. Keep in mind that Regnum follows strict protocols and has a very intense quality assurance program to verify compliance with its protocols in order to keep FDA approval.”
All three of us groaned at his defense of his former employer.
“Look, they are not a bad company.”
“They just made some bad decisions,” I said.
“And they are going to pay for it,” Neil said. “I suspect that if you are right about where it came from, they will pay a lot. I’ll have to think about the possibilities, but I wouldn’t rule out something intentional.”
“You serious?” I asked. “Could someone possibly have designed this just for kicks?”
“I’m not ruling anything out. My brain is a bit jumbled, but after some sleep I’ll try to walk through each of the steps and think about the possible points of entry. I’ll have to think about each of the people that I knew there and whether any of them would have A) been capable and B) been motivated to do this.”
“So how do we bring this to Regnum so that they listen and take the right action, namely pull the vaccine right away?” I asked. “I suspect that once they learn about this they will start building some mighty big walls.”
“You can be sure of that,” Neil acknowledged. “This is going to take some
finesse. Who else knows about this? Have you told Johnathan Oros?”
“I think we need to leave him out,” I said. I know you brought him in at the beginning and he has offered some funding, but as of yet, he hasn’t been involved at all, and based on Regnum’s vaccine being the cause, his lab won’t be needed.”
“Your call, I just want to make sure that when the press finds out, that—I just think it is critical that we are all on the same page. Claire, you should be the spokesperson, and everyone should refer all questions to you.” He looked at Anna and she nodded in the affirmative.
“Actually, I’m going to have to coordinate with the university communications office, and probably the legal office, and—this is going to be way bigger than any of us have really allowed ourselves to think about.” I shook my head to shake out the complexities.
Chris shouldered me. “It’s going to be huge.” He shook me gently. “Neil is right, you have to make sure everyone is of a single mind in how this will be addressed publicly and with Regnum. Everyone has to be committed to one goal.”
“Stopping the dog deaths,” Anna said, finally looking a bit weary.
Neil put his arm around her. “We will. You all have found the culprit, now we need to bring this to an end, quickly.”
“And hope that the folks at Regnum are as committed,” I said.
We all looked at Neil.
He nodded hopefully. “Ending this is—”
Anna and I alternated. “Simple.”
“Stop using the vaccine.”
“Millions of dollars, millions of research hours,” Chris said.
“And thousands of dogs,” Anna added
“I get it,” Neil acknowledged. “The solution is straightforward, but not really simple. I take it that you all grasp the implications?”
“We grasp the implications for you, yes,” Anna said, and Neil shook his head no, not a concern. “And for Regnum. But mostly we grasp the implications if this continues even one more day. I’m concerned that with prolonged exposures that sooner or later some dogs are going to show some immunity, and may become carriers, and we will have a real epidemic on our hands. There are millions of dogs out there.”
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