Torturing more mice in the name of antivaccine pseudoscience

Of all the vaccines out there, it’s hard for me to decide which among them antivaccine activists fear and detest the most. Sure, there’s the MMR vaccine, the original granddaddy bete noire, demonized so successfully by Andrew Wakefield as causing autism based on some of the flimsiest evidence ever, evidence later shown to be fraudulent. That has to be near the top of the list of any of the vaccines demonized by the antivaccine movement, despite is safety and efficacy. After all, it is the mMR vaccine that people like Del Bigtree, Andrew Wakefield, and Polly Tommey are still flogging as the cause of autism, spinning their very own conspiracy theory about it known as the “CDC whistleblower,” in which a CDC scientists, William Thompson, has been portrayed as having claimed that the CDC covered up evidence that the MMR was associated with nearly a four-fold increase in the risk of autism in African-American males. They even have a movie, VAXXED, about it, a propaganda film disguised as a documentary.

However, if you want real fear and loathing, look no further than HPV vaccines, such as Gardasil or Cervarix (particularly Gardasil in this country). HPV vaccines have been blamed for everything from premature ovarian failure to even death, as well as the contamination of our precious girls’ precious bodily fluids with the dreaded HPV DNA. Never mind that it’s a safe and effective vaccine that prevents infection with the most common strains of HPV that lead to cervical cancer. Or maybe part of it is because exactly that, as there seems to be an almost willful denial that my girl would ever need a vaccine to protect her against a sexually-transmitted virus, because my girl is a “good” girl and would never (apparently) engage in premarital sex or have more than one sexual partner in her lifetime.

None of that was enough apparently. Just as antivaccinationists like Yehuda Shoenfeld make up fake “syndromes” like Autoimmune/Inflammatory Syndrome Induced by Adjuvants (ASIA) for other vaccines and blame other vaccines for behavioral problems based on shoddy mouse experiments retracted and republished, so too, apparently, antivaccinationists need to make up a new syndrome of its very own for HPV vaccines, this time calling it “HPV vaccination associated neuro-immunopathetic syndrome” (HANS), but, as you will see, in reality it’s just ASIA with another name and just as convincing—as in not very.

I first learned of HANS in a journal article circulating among the antivaccine underground. It was published in Scientific Reports, which is apparently Nature’s answer to PLoS One, but in reality is increasingly striking me as Nature’s answer to Medical Hypotheses, the journal long famous for publishing “speculative” papers on how vaccines cause autism and a number of other scientifically dubious topics. Am I being harsh? Maybe. It could be confirmation bias. But I’m increasingly getting the impression that Scientific Reports is the new go-to journal for pseudoscience. Be that as it may, Scientific Reports shares the same criteria as PLoS One for publication, namely that a paper doesn’t have to have high impact or importance. It just has to be scientifically valid. Indeed, Scientific Reports is on track to surpass PLoS One as the largest scientific journal in the world.

Open access journals are a mixed bag. PLoS One, for instance, has published some really good science, but it’s also published some truly terrible science. A skeptical eye is required. This article in Scientific Reports is as bad or sorse than anything I’ve seen in PLoS One—or anywhere else, for that matter. One reason I mention the journal is because of the confusion. Because Scientific Reports is published by Nature Publishing Group (NPG), articles published there are sometimes mistakenly viewed as having been published in Nature or by Nature. Given that Nature is one of the top three or so scientific journals in the world, that mistake can lead articles appearing in Scientific Reports to be viewed as far more prestigious than they should be. I can’t help but wonder if NPG knows this and uses this and that’s why Scientific Reports is growing so fast.

On to the paper, which is authored by Aratani et al and entitled Murine hypothalamic destruction with vascular cell apoptosis subsequent to combined administration of human papilloma virus vaccine and pertussis toxin. Now, my first thought upon reading the title was: Why on earth were the investigators combining pertussis toxin with HPV in mice? It couldn’t be that they wanted to make sure they saw an inflammatory reaction, could it? Perish the thought! Of course, As Yehuda Shoenfeld has done for ASIA, Aratani et al do for HANS. They assume the existence of facts not in evidence, namely the existence of HANS as an actual syndrome:

To evoke a full- and adequate immunological reaction, vaccines include aluminum adjuvant in addition to virus-like particles as antigens. Chronic stimulation of the immune system by adjuvants can result in an autoimmune disease characterized by myalgia, arthralgia, chronic fatigue, and neurological manifestations – appropriately named the autoimmune/inflammatory syndrome induced by adjuvants (ASIA)^7,8. According to the HPV vaccination, there are two recent well-documented review articles. Palmieri B. et al reported the occurrence of severe somatoform and dysautonomic syndromes after HPV vaccination⁹ and Brinth L. et al. also described the onset of autonomic dysfunction after the quadrivalent vaccination¹⁰. Both reviews clearly indicated the presence of unique adverse reactions associated with the HPV vaccination including headache, fatigue, depression, cognitive dysfunctions, uncontrollable and involuntary movement, and limb weakness. For these clinical manifestations, we have coined these reactions as human papillomavirus vaccination-associated neuro-immunopathic syndrome (HANS) and proposed diagnostic criteria. HANS syndrome is thought to consist of four clinical domains; (i) autonomic, endocrine and inflammatory symptoms; (ii) cognitive and emotional symptoms; (iii) environmental hypersensitivity and pain symptoms and (iv) locomotion and motor symptoms11,12. Several clinical studies on HANS symptoms have also shown that the HPV vaccines may influence the central nervous system (CNS)^10,13,14,15.

Let’s take a look at one of these reviews. The first, by Palmieri et al, Severe somatoform and dysautonomic syndromes after HPV vaccination: case series and review of literature, is a retrospective case series of patients referred to something called the “Second Opinion Medical Network” at the University of Modena and Reggio Emilia Medical School in Modena, Italy. Sadly, this appears to be a general surgery clinic. It was a small case study of 18 girls, all of whom complained of “long-lasting and invalidating somatoform symptoms (including asthenia, headache, cognitive dysfunctions, myalgia, sinus tachycardia and skin rashes) that have developed 1–5 days (n = 11), 5–15 days (n = 5) and 15–20 days (n = 2) after the vaccination.” The authors then catalog the complaints but don’t do much in the way of further investigation, other than to strongly imply that correlation equals causation and to speculate that the girls’ symptoms fit in with ASIA, which, of course, is so protean in its manifestations that just about any set of vague symptoms can be attributed to ASIA. Unfortunately, this article was credulously reported on Medscape, which so bought into the unwarranted speculation that it drew a rebuke from Paul Offit, who noted:

First of all, the HPV vaccine was studied for safety in 30,000 people for 7 years before licensure.[2] It has been formally studied both in phase 4 postlicensure studies and by the Vaccine Safety Datalink in more than a million people, and has been found not to cause chronic fatigue or fibromyalgia. When those symptoms do occur, they occur at the same rate in both vaccinated and unvaccinated groups.[3]

We learned from these studies that the HPV vaccine doesn’t prevent fibromyalgia or chronic fatigue in adolescents. That Medscape chose to highlight this article,[1] as if it were in any way an advance, is disappointing. Frankly, this falls under the same category as the syndrome described by Andrew Wakefield—that the MMR vaccine caused intestinal symptoms and autism, which also was thoroughly debunked.[4]

Yep. This “study” is nothing more than some cherry picked cases—the investigators were actually looking for girls complaining about these sorts of symptoms after vaccination—combined with confusing correlation with causation and speculation about an as yet unproven clinical entity.

But what about the mouse study? Whenever I see a study like this, one of the first questions that come to mind is: Where the hell was IACUC, or, in this case, the Japanese equivalent of IACUC? IACUC, for those not familiar with it, is the committee at each university and institution charged with protecting animal welfare, assuring that animal research is conducted ethically and humanely, and following federal and state laws and regulations governing animal research.

It wasn’t long before I learned why Aratani et al administered pertussis toxin in addition to Gardasil:

To assess the neurophysiological effects of the HPV vaccine, forty eight female mice were either immunized with Gardasil or given phosphate-buffered saline (PBS) as a control (Fig. 1-a). Pertussis toxin (Ptx) was administrated subsequent to the vaccination to facilitate the access of the vaccine to the CNS via modulation of the blood-brain barrier (BBB)¹⁶.To assess the neurophysiological effects of the HPV vaccine, forty eight female mice were either immunized with Gardasil or given phosphate-buffered saline (PBS) as a control (Fig. 1-a). Pertussis toxin (Ptx) was administrated subsequent to the vaccination to facilitate the access of the vaccine to the CNS via modulation of the blood-brain barrier (BBB)16. As a control for autoimmune encephalomyelitis, myelin oligodendrocyte glycoprotein (MOG)^35-55 was used¹⁷.

So this is a totally artificial system. The pertussis toxin is intended to cause “leakiness” of the blood-brain barrier, which would normally keep out proteins like the ones in Gardasil. As a positive control, they injected MOG to induce autoimmune encephalitis. The experimental groups were thus:

1. Vehicle (phosphate-buffered saline, or PBS)
2. Pertussis toxin + PBS
3. Gardasil + PBS
4. Gardasil + pertussis toxin
5. MOG + PBS
6. MOG + Pertussis toxin

The first thing that struck me about this paper is the paucity of quantitative data and the utter lack of any sort of statistical analysis. Nor is there any indications that the observers who observed neurologic symptoms, such as tail drop, and scored the disease severity using various instruments were blinded to experimental group. Ditto the investigators who scored the tissue sections for immunohistochemistry (IHC). There’s a nontrivial amount of subjectivity in scoring IHC, as all pathologists know. To me, any study that does IHC studies in which the pathologist or investigator scoring the tissue sections isn’t blinded to treatment group deserves extreme skepticism because such measurements are extremely prone to subtle unconscious bias that can affect choice of areas examined and how cells that stain weakly are called (positive or negative). After all, if you look at the supplemental data section, you’ll see that there was no difference between the mice in the vehicle control group and the Gardasil + pertussis toxin groups in the mobility of the mice as measured by the horizontal bar test and Kondziela’s inverted screen test.

So basically, the investigators report that mice inoculated with HPV and pertussis toxin had impaired responsiveness of the tail reflex and reduced locomotive activity. Again, this is a very artificial system basically designed to show something. Pathological analyses supposedly revealed damage to areas of the brain (the hypothalamus and circumventricular regions) around the third ventricle, as well as increased apoptosis (programmed cell death) in the vascular endothelial cells (the cells lining the blood vessels) in these regions.

One commenter noted exactly this problem and others that I’ve mentioned above:

If seven times more apoptotic cells were observed in mice treated with Gardasil and pertussis toxin than those in control mice, how much were the actual number (e.g. the average number per 10 or more high power fields)? Similarly, if tyrosine hydroxylase was increased in PVN, and glutamic acid decarboxylase 67 and gamma-aminobutyric acid was decreased in PVN, how did they assess these findings (e.g. by comparing the intensity of positive staining per 10 or more high power fields)? They did not mention at all how they analyzed these pathological findings. Instead, they only showed numerous microscopic pictures which might manipulate readers’ images.

If they had assessed them only by their subjective impressions, it would be highly vulnerable to confirmation bias or even intentional overestimation, unless the microscopic examiners had been blinded about whether each specimen was derived from a vaccinated or non-vaccinated mouse. Otherwise, it might be reasonable to request some researchers of other institutes, who are blinded about the information of those specimens, to retest the pathological analysis. If such a retest should corroborate their conclusion, their study would obtain a robust credibility, and in turn, further evaluation of the safety of HPV vaccines might be necessary.

As for risk of confirmation bias, it is all the same for the neurophysiological
tests for the mice. Their behavioral tests for the mice revealed no significant differences between the vaccinated mice and the control.

Exactly. This commenter also noted that the authors never really defined a lot of rather important criteria for their IHC analysis in terms of neuroanatomy and how they picked their sections to examine. Again, when the authors are unblinded, unconscious bias can easily affect selection of anatomic areas. That’s why we blind researchers in clinical research. Unfortunately, researchers in basic research often seem not to understand how important blinding is when it comes to measurements like this. Basically, this is an utterly useless paper, a waste of precious animals. Worse, it’s animal torture—and I mean that literally—for no good scientific reason.

Antivaccinationists like to postulate the existence of autoimmune diseases and syndromes that arise as a result of vaccines, basing their ideas on the extremely rare incidence of vaccine complications such as Guillan-Barre syndrome, a syndrome so rare that it’s still not entirely clear that it’s linked to vaccines. They invent syndromes like ASIA, and now HANS (which, let’s face it, is basically just ASIA with a different name). Neither condition has been demonstrated to exist.

This brings me back to Scientific Reports. I’ve always supported the existence of open access journals like PLoS One (and now Scientific Reports) dedicated to publishing scientifically valid research that might not be as impactful as what other journals publish or that might not (yet) make a complete story (although, having published a couple of times in PLoS One, I can’t help but notice that its review process has become very much like that of the other journals I’ve published in). However, whatever Scientific Reports means by “valid,” this paper by Palmieri et al fails that test. To me “valid” scientific research means proper experimental design, including blinding, selection of measurement criteria, and proper quantitative and statistical analysis, as well as sufficiently detailed reporting to allow other investigators to know what was done and evaluate the results. This paper fails both criteria massively.

NPG has a great reputation, thanks to its flagship journal Nature. Publishing crap like this on Scientific Reports risks both that reputation, but, worse, can easily allow cranks and quacks to falsely but convincingly claim that Nature published putridly awful research like this supporting their ideas.