Tuesday, 13 August 2013

It's a Monkey. It's a Cow. It's a Pig... It's Rotavirus!

Let's take a look at this expensive optional Rotavirus vaccine that is recommended for infants between 6 weeks to 6 months old, with 2 doses for Rotarix or 3 doses for RotaTeq (at least 4 weeks apart). As we examine the necessity for vaccines, we need to understand the disease and how it can be treated or prevented.

  • Rotavirus is the most common cause of diarrhea among infants.
  • Rotavirus causes fever, vomiting, and diarrhea.
  • Rotavirus spreads through the oral-fecal route via contaminated hands and surfaces
  • Rotavirus typically resolves on its own within 3 - 9 days.
  • Two Rotavirus vaccines are available in Malaysia. Rotarix (GSK) since July 2006 and RotaTeq (Merck) since July 2007 [11]. 
  • Rotavirus vaccine is a live, oral vaccine.
  • Rotavirus vaccine is not recommended for infants above 1 year old. Why? Older children very often already had a rotavirus infection, so there's no point in vaccinating them [27]. Also, as they get older, some babies – about one in a thousand – get a condition that causes a blockage in their lower gut (intussusception). Most cases happen between 5 months and a year. Therefore, vaccination should not be initiated for infants age 15 weeks 0 days or older because there are insufficient data on the safety of dose #1 in older infants [27, 28]. 
Why is the rotavirus vaccine recommended?
The main reason is that severe dehydration can be dangerous for infants. Dehydration happens when the infant isn't drinking enough water, milk, or fluids. 

What's the treatment for rotavirus?
Plenty of fluids such as breastmilk, water, coconut water, barley water.

How to prevent rotavirus?
Proper hygiene by washing hands, taking bath and cleaning surfaces. If any member of the family has been infected with rotavirus, it is important to have proper disposal of used diapers. Environmental surfaces should be cleaned with soap and water, and disinfected.

Now that we have listed out the facts, what are the possible side effects for the rotavirus vaccine? Among the common side effects are fever, vomiting and diarrhea. Yes, the exact same symptoms of the disease along with a hefty pricetag. Oh yes, that doesn't include the rare risk of intussusception.

Anyway, intussusception doesn't seem to be very rare when there are over 100 cases of it.
By December 2000, VAERS had received over 100 reports of confirmed intussusception cases, 58 of which had onset within 7 days of vaccine receipt. Of the cases reported, approximately one-half required surgical intervention. Nearly all of the other cases of bowel obstruction were relieved through barium enema.
In February 2007, the FDA notified health care providers and consumers about 28 post-marketing reports of intussusception following administration of RotaTeq.
A 2011 post-marketing study of RotaTeq published in "Vaccine,'' from the Australian National Immunization Program, suggests an association between RotaTeq and intussusception. Approximately 295,000 doses of RotaTeq were given in two states. When the 1-21 day interval following the first dose was examined, six cases of intussusception were found. [9]

It's a Monkey. It's a Cow. It's a Pig... It's Rotavirus!
Ah, now to the juicy portion of this post. You might be wondering where did the monkey, cow and pig join the rotavirus vaccine party. The Rotavirus vaccine is cultured using viral seed stock. A seed culture is the first culture used to start stock cultures. 

History of Rotarix
In the development of the vaccine, the virus needs to be attenuated (weakened). There is no adjustable knob for how strong or weak we'd like the vaccine to be. If the virus is used without being weakened, the result would be a full-blown rotavirus infection. Therefore, the live virus is weakened by transferring or passaging it through different host's cells. The Rotarix vaccine is passaged through the African Green monkey kidney cells. This monkey is famous for the cancer-causing Simian Virus 40 (SV40) contamination in the oral polio vaccine [13]. Anyway, not to worry, since the SV40 contamination was identified in 1960 after more than 10,000 Americans and millions in the USSR where clinical trials were conducted, the Rotavirus vaccine should be SV-40 free. Table 1 shows the routine manufacturing process for Rotarix (GSK). 
The Serotype G1 HRV strain (genotype P[8]) which GSK used to make vaccine product is designated RIX4414. It was derived from strain 89-12, initially developed by Avant Therapeutics, Inc. ---------------. The virus was isolated in ------------- from a child in Cincinnati with a natural case of rotavirus with mild diarrhea. This original isolate was passaged 26 times in primary African Green monkey kidney cells (AGMK) by Avant for use as seed material. The P26 virus was ------- passaged by -- --------------- AVANT, -------, which passaged the seed virus an additional 7 passages to P33. This was the material that was clinically tested ---------------. The additional 7 passages were performed in an AGMK cell line that ------- characterized in --------.[3]
First, we start with the monkey component of this vaccine. To grow the virus culture, we need a cell medium. Vero cells are derived from the kidney of an African green monkey (Cercopithecus aethiops) in the 1960s, Vero cells are one of the most common mammalian continuous cell lines used in research because it is susceptible to a broad range of viruses [16].

Here's where the cow joins the party. Bovine serum was included in the culture medium used for adaptation of the Vero cells to serum-free culture conditions (first passage from the master cell bank) [14]. Donor calf serum is collected from bovine calves less than eight months of age from bovine blood collected in USDA-inspected abattoirs located in the United States (it doesn't specify how it is slaughtered) [23, 24].

No materials of bovine origin are used in the current routine production process of Rotavirus vaccine, including the master seed (MS RVCL13E02) and the current working seed (RVCL29G02), except for lactose (from bovine milk fit for human consumption) which is used as a stabiliser for porcine trypsin [14].

Table 1: The Rotarix vaccine manufacturing process. [2]

Trypsin for the rotavirus vaccine is the pancreatic enzyme of a pig from USA [14]. Trypsin is added in the manufacturing process.for cell passaging. The viral seed is usually cultured and grown in petri dishes, multi-wells plates, or culture flasks. When the food runs out and nothing is done, the cells will die shortly thereafter, so passaging is required and trypsin is utilized to make the cells detach from the bottom of the plate [5].

Table 2: How is trypsin prepared. [2]

The Rotavirus vaccine is contaminated at every level of the manufacturing step with porcine circovirus since the seed culture is contaminated (Table 3).

Table 3: Rotarix vaccine shows Positive result for contamination with porcine circovirus (PCV). [2]

No studies on animals have been done to study the potential for hypersensitivity and autoimmune reactions Even large-scale clinical trials cannot prove that a vaccine is completely safe in this respect. Ultimately, post marketing surveillance studies are the only tool to study whether a new vaccine can cause autoimmune disease or allergic reactions [14]. Ah, so they need to use human guinea pigs to find out if the vaccine may trigger allergies or autoimmune diseases!

History of RotaTeq
Rotateq virus seed lot was produced by mixing bovine and human rotavirus. It uses the Vero cell line from the kidney of the African green monkey in 1962. During the vaccine bulk manufacturing process, porcine trypsin solution is added to ensure efficient cleavage of the VP4 spike; cleavage of this viral protein is necessary for infection. Raw materials of animal origin are present in the culture media, cell substrates, and virus seeds used to produce the drug substance, final formulated bulks (FFB), and the drug product. The animal-derived raw materials include: fetal bovine serum, porcine pancreatic trypsin solutions, cholesterol, polysorbate-80 (PS-80), and amino acids. Bovine-derived PS-80 was used to manufacture seeds and early clinical  lots; however, plant-derived PS-80 is currently used in the manufacturing process. [25]

Fetal bovine serum (FBS) is a common component of animal cell culture media. It is harvested from bovine fetuses taken from pregnant cows during slaughter. FBS is commonly harvested by means of a cardiac puncture without any form of anaesthesia. Fetuses are probably exposed to pain and/or discomfort, so the current practice of fetal blood harvesting is inhumane [26]. 

The differences between Rotarix, Rotateq and RotaShield [8]:
  • Rotarix is composed of a (monovalent) human attenuated rotavirus strain (derived from a human strain, "89-12", which belongs to the G1 serotype and P[8] genotype). This is a single strain vaccine so it is not effective against other strains of rotavirus. There may be more than 80 strains around and it varies from one geographic region to another, and even year to year [18].
  • RotaTeq is a "pentavalent" vaccine, composed of five reassortants rotavirus strains derived from a bovine strain (each of which contains a gene encoding VP4 or VP7 from human origin). This is a genetically engineered vaccine whereby 5 strains of human and cow strains are cross bred [17].
  • RotaShield (which was withdrawn from the market in 1999) is composed of 4 human-simian reassortants containing four serotypically distinct VP7 components.
Rotavirus vaccine ingredients.
Rotavirus (RotaTeq) - sucrose, sodium citrate, sodium phosphate monobasic monohydrate, sodium hydroxide, polysorbate 80, cell culture media, fetal bovine serum, vero cells [DNA from porcine circoviruses (PCV) 1 and 2 has been detected in RotaTeq. PCV-1 and PCV-2 are not known to cause disease in humans.]
Rotavirus (Rotarix) - amino acids, dextran, , sorbitol, sucrose, calcium carbonate, xanthan, Dulbecco’s Modified Eagle Medium (DMEM) [Porcine circovirus type 1 (PCV-1) is present in Rotarix. PCV-1 is not known to cause disease in humans.] [10]

Ah, now it makes perfect sense why the virus culture needs to undergo testing for adventitious viruses and is subject to a contamination issue that is still unresolved. Anyway, the vaccine manufacturer assures us that the vaccine is perfectly safe since there is no evidence (yet) of human infants catching PCV1 and PCV2 from the vaccine. Didn't they also assure us when they released the vaccine that it had passed all safety standards? Oops, and then they suspended it in March 2010 due to PCV contamination, and then they added intussusception as a contraindication in 2011. 

It was a bit difficult to find a recent research paper that is not influenced by the rotavirus vaccine hype. This is a research article that was conducted before the rotavirus vaccine was introduced in Malaysia. According to this study in University of Malaya Medical Centre, Kuala Lumpur, Malaysia, between 1996 and 1999; 333 cases (24%) of 1362 stool samples, obtained from children admitted with acute diarrhoea, were positive for rotavirus. No deaths were recorded [19]. Another study of all patients younger than 12 years of age admitted with acute gastroenteritis to Queen Elizabeth Hospital, Sabah, over a six-month period (October 2005 to March 2006)  examined a total of 167 stool samples from children with acute gastroenteritis. Rotavirus accounted for 16% of all diarrhoea cases, and 1.7% of all admissions [21].

We can summarize that less than a quarter of diarrhoea cases admitted to these 2 hospitals in Malaysia are caused by a rotavirus infection. It would be interesting to find out which rotavirus strain caused the disease in these patients. For those planning to take the rotavirus vaccine, it might be prudent to have an allergy test beforehand, since any sensitivity to the vaccine or its components are a contraindication.

Food for thought: Does my child need a contaminated vaccine along with the risks of vaccine side effects? Is it safer to ensure my child practices good hygiene and consume sufficient fluids in times of illness?

Definition of terms
Bovine - Cow origin
Porcine - Pig origin
Simian - Monkey origin
Adventitious viruses - A contaminant virus present by chance in a virus preparation. [7]

  1. Rotavirus infections http://www.childrenshospital.org/az/Site1533/mainpageS1533P0.html
  2. PCV-1 detection in Rotavirus vaccine: A case study in the risk assessment for adventitious agent testing (GSK) http://www.iabs.org/index.php/docs/doc_download/124-pcv-1-detection-in-rotavirus-vaccine-a-case-study-in-the-risk-assessment-for-adventitious-agent-tes
  3. Review of Vero Cell Banks used for Vaccine Production and Adventitious Agent Testing of Virus Seeds and Vaccine Human Rotavirus Vaccine (HRV) - Rotarix http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm134138.htm
  4. Guidelines to assure the quality, safety and efficacy of live attenuated rotavirus vaccines (oral) http://www.who.int/biologicals/publications/trs/areas/vaccines/rotavirus/Annex%203%20rotavirus%20vaccines.pdf
  5. Cell Passage and the Use of Trypsin http://www.carlroth.com/website/fr-fr/pdf/Zellpassage_Trypsin_E.pdf
  6. Adventitious viruses http://www.answers.com/topic/adventitious-virus
  7. Rotavirus vaccine package inserts http://www.immunize.org/packageinserts/pi_rotavirus.asp
  8. Review Memo - CMC Section - Rotarix http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm133612.htm
  9. National Vaccine Injury Compensation Program http://www.gpo.gov/fdsys/pkg/FR-2013-07-24/html/2013-17786.htm
  10. Vaccine Excipient & Media Summary http://www.cdc.gov/vaccines/pubs/pinkbook/downloads/appendices/B/excipient-table-2.pdf
  11. Giak CL, Singh HA, Nallusamy R, Leong TY, Ng TL, Bock HL (2008). Epidemiology of Intussusception in Malaysia: A Three Year Review. http://www.tm.mahidol.ac.th/seameo/2008_39_5/12-4356.pdf
  12. Intussusception Currant Jelly Stool http://jjmedicalschool.blogspot.com/2009/04/currant-jelly-stool.html
  13. Simian Virus (SV40) A Cancer Causing Monkey Virus from FDA-Approved Vaccines http://www.sv40foundation.org/CPV-link.html
  14. EME Scientific Discussion http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Scientific_Discussion/human/000639/WC500054589.pdf
  15. Growth and Maintenance of Vero Cells http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657228/
  16. What are Vero Cells? http://biotech.about.com/od/faq/f/VeroCells.htm
  17. Second Rotavirus Vaccine Now Available http://www.askdrsears.com/topics/vaccines/second-rotavirus-vaccine-now-available-and-timing-doses-has-been-expanded
  18. Rotavirus strains http://explorevaccines.wordpress.com/2011/04/03/rotavirus-strains/
  19. Lee WS, Veerasingam PD, Goh AY, Chua KB (2003). Hospitalization of childhood rotavirus infection from Kuala Lumpur, Malaysia. Journal of Paediatric Child Health, 39(7), 518-522. http://www.ncbi.nlm.nih.gov/pubmed/12969206
  20. Goh CT, Cheah PK, Soo TL, Lee WS (2009). The epidemiology and burden of childhood rotavirus infection in a tertiary hospital in Sabah, Malaysia. Medical Journal of Malaysia, 64(2), 146-149. http://www.ncbi.nlm.nih.gov/pubmed/20058575
  21. Goh CT, Cheah PK, Soo TL, Lee WS (2009). The epidemiology and burden of childhood rotavirus infection in a tertiary hospital in Sabah, Malaysia. Medical Journal of Malaysia, 64(2), 146-149. http://www.ncbi.nlm.nih.gov/pubmed/20058575
  22. Donor Calf Serum http://cellgro.com/products/serum/animal-sera/donor-calf-serum-u-s-sourced.html
  23. Calf Bovine Serum http://www.atcc.org/products/all/30-2030.aspx
  24. EMA Rotateq http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Scientific_Discussion/human/000669/WC500054183.pdf
  25. The use of fetal bovine serum: ethical or scientific problem? http://www.ncbi.nlm.nih.gov/pubmed/11971757 
  26. Rotavirus - Ask the Experts http://www.immunize.org/askexperts/experts_rota.asp
  27. Rotavirus - Frequently Asked Questions http://www.nhs.uk/Conditions/vaccinations/Pages/rotavirus-vaccine-questions-answers.aspx