Bovine Spongiform Encephalopathy (BSE)

Estimating Risks for vCJD in Vaccines Using Bovine-Derived Materials

The risk of vCJD from bovine-derived materials

The risk of developing an illness such as vCJD from the use of bovine-derived material in the manufacture of vaccines is a function of a number of factors, including the nature and the amount of the bovine tissue that is used in manufacture, as well as the date and country of origin of the cows (1). Other factors, such as how the cows were fed, are also important. In this regard, the CDC estimates that the risk, if any, for vCJD from eating a beef meal in Europe is less than approximately 1 in 10 billion [http://www.cdc.gov/travel/madcow.htm].

CBER's survey of vaccine manufacturers revealed a number of vaccines that utilized bovine materials that were obtained from countries where BSE or a significant risk for BSE exists. An estimate of the risk that the use of these materials might pose is presented in the following sections. Two examples have been chosen for presentation here, namely, the risk from the use of fetal calf serum sourced from the United Kingdom (UK) in the derivation of a viral working seed that is subsequently used in vaccine manufacture and the use of European-sourced (excluding the UK) beef broth in the production of a bacterial toxoid. Based on CBER's survey of the use of bovine-derived materials sourced from countries on the USDA BSE-list, the potential risk that would be associated with other uses of bovine-derived materials in vaccine production would be less than might be associated with these two situations.

The infectivity of most bovine-derived materials has not been determined experimentally. More is known about the infectivity of various ovine-derived (from sheep) materials. The knowledge of the infectivities of different ovine tissues relative to each other can be used to estimate the relative infectivities of bovine tissues. For example, if we know that, on a gram for gram basis, sheep brain is 100 million times more infective than sheep muscle, we can assume that bovine brain is also 100 million times more infective than bovine muscle. Thus, if the infectivity of bovine brain has been measured, and contains 10 million infective doses per gram, then we can estimate that bovine muscle is 100 million times less infective and contains 0.1 infective dose per gram.

Not surprisingly, since BSE and scrapie (the corresponding disease in sheep) are neural diseases, the greatest infectivity is found in neural tissue. Based on experimental studies, infected bovine brain contains approximately ten million infectious units per gram when administered to other cattle (2,3). In other tissues, such as serum or skeletal muscle, no infectivity has been detected. This does not mean that there is no infectivity associated with these materials; only that, if they are infectious, then the infectivity is at a level that is too low to be measured by current tests.

Table I presents the estimated infectivity of different bovine-derived tissues as determined by The European Agency for the Evaluation of Medicinal Products (EMEA)­ The actual infectivity of skeletal muscle or serum, for example, may be well below the values shown; we will, nevertheless, use these values in our risk estimates. It should be noted that these values are based on experiments in which animals were infected by intra-cerebral injection with affected tissue; this is the most effective means of infecting experimental animals. When another route of administration, namely intramuscular injection, is used, infection rates are estimated to be approximately 200 fold lower (4).

The risk assessments follow.

Fetal calf serum used to derive viral seed and cell banks

Fetal calf serum from the United Kingdom was used in the production of certain viral seeds and cell banks. The calf serum that was used was produced in the mid-1980s, when the BSE epidemic was just getting underway in the UK (5). The U.S. Department of Agriculture estimated the incidence of BSE in adult cattle at about 1 in 200 at that time(6). [Although many fewer cattle were observed to suffer from mad cow disease at that time, the long incubation time for the disease means that more cattle were infected than appeared diseased.] Since fetal calf serum was used in the production of the cell and viral seed banks, it is necessary to address the question of maternal-fetal transmission. Whether there is mother to fetus transmission of BSE is still unknown. One study may be interpreted as indicating that maternal-fetal transmission occurs at a rate of approximately 10%; i.e., that the calves of one of ten infected mothers may become infected with the BSE agent (7). However, other data indicate that maternal-fetal transmission does not occur or, if it does occur, it is below this 10% rate (8). As noted above, the U.S. Department of Agriculture estimates that, during the mid 1980s, approximately 1 in 200 cows in the United Kingdom was infected with BSE. Assuming that the rate of transmission from mother to fetus is 10% we would then estimate that 1 in 2000 fetal calves would have been infected.

When fetal calf serum is manufactured, the sera from approximately 1500 calves are pooled together. If 1 in 2000 calves is infected, it is likely that any given serum pool is infected. As mentioned above, although no infectivity has been observed with serum, there are limits to detectability. These experiments only rule out an infectivity that is greater than 1 infectious unit per milliliter (mL) of blood (3,9,10). Although serum is listed as category IV, we are using the highest estimate consistent with infectivity experiments. In the following risk estimate, we assume that the serum of an infected fetal calf can contain up to 1 infectious unit per mL.

In our risk calculation, we assume that the number of infectious BSE units that enters the vaccine production process is equal to the number of infectious units that remain in the vaccine at the end; that is, that the risk for vCJD is the input number of infectious units divided by the number of doses of vaccine that is in the batch. Thus, the risk estimate does not account for any purification step that might be present in the viral vaccine manufacturing process; although there are steps that probably remove infectivity, these are not considered in our risk estimate since none of the manufacturing steps have been demonstrated to remove BSE infectivity. We have also assumed that the BSE agent does not replicate during the manufacturing process; this is a reasonable assumption, bolstered by the many failed attempts to propagate the BSE agent in cell culture (11). The BSE infectivities that are estimated in Table I are derived from data using direct intra-cerebral inoculation (direct injection of the material into the brain). Vaccines are given intramuscularly, a less efficient route of transmitting the disease. In our risk estimate, we have allowed a factor of 200 for reduced transmission by the intramuscular route.

In general, there is a species barrier for the transmissible spongiform encephalopathies; that is, it is easier to infect the same species of animal than another species (for example, bovine material is more infectious for cows than it is for other animals, such as mice) (3,4). The species barrier from cows to humans is not known; in our calculations, we will therefore assume that there is none.

Given these assumptions, we can estimate the risk for vCJD from fetal calf serum (FCS) being used to prepare a viral working seed as the product of four separate risk factors. The level of BSE agent in the serum of an infected calf is estimated at 1 infectious unit per mL. Approximately 1 infected calf is present in each pool, deriving from approximately 1500 calves, of fetal calf serum. The infectivity of the pooled FCS is thus diluted to 1/1500 infectious units per mL (ca. 6.7 x 10-4 infectious units/mL). The amount of FCS that was used to produce a vial of a working viral seed is approximately 4 mL, and the number of doses of vaccine coming from that batch is approximately 500,000. The risk for acquiring vCJD is therefore:

The number of infected calves in each pool 1/1500
       Multiplied by
The number of infectious units per mL of serum 1
       Multiplied by
The number of mLs of serum used 4
       Divided by
The number of doses of vaccine 500,000
       Divided by
The reduction in infectivity related to the route of administration 200

This yields a final risk estimate for vCJD of approximately 2.5 per 100 billion or 1 in 40 billion doses of vaccine [(1/1500) x 1 x 4 x (1/500,000) x (1/200)]. This level of risk would correspond to one case of vCJD arising every 5,000 years (assuming two doses per child) when vaccinating the entire birth cohort of the Unites States (four million children). Because of the assumptions that were used, this is an overestimate of the risk, and the true risk is likely to be significantly less. The risk that would be calculated for the use of a master seed that was prepared with fetal calf serum is again considerably less, due to an additional dilution that attends the preparation of the working seed from the master seed.

Beef broth used to manufacture a bacterial vaccine: a bacterial toxoid as an example

The potential risk of vCJD from a bacterial vaccine that used bovine-derived material in the nutrient broth to grow the bacterial strain during vaccine production is as follows. In the example that we are using, tissue derived from a single cow is used to prepare the fermentation broth. For this estimate, the incidence of BSE in European cows is taken to be 1 in 10,000. This value was derived by multiplying the average BSE rate in this region over the last five years by a factor of ten (1) to account for any uncertainty in the actual rates. The nutrient medium that is used to grow the bacteria for the vaccine contains approximately 750 grams of skeletal muscle (a Category IV material) and 200 grams of a pancreatic extract (a Category III material); see Table I. Because the broth is autoclaved (heated at high temperature), some of its potential infectivity is lost; a reduction factor of 20 is assigned to the autoclaving process(2).

The risk, per dose of vaccine, for vCJD from a vaccine using a beef/pancreatic extract can be calculated as the product of the risk of using an infected cow (1 in 10,000) times the inherent risk of the bovine material after correction for the autoclaving process (approximately 1000 units; [200 grams of Category III material is estimated to contain no more than 20,000 infectious units and the 750 grams of Category IV material no more than 75 infectious units (20, 075 units total); the autoclaving process reduces this infectivity to approximately 1000 units]), divided by the number of doses that are in a batch of vaccine (approximately 1 million), corrected for the route of administration (a reduction factor of 200).

Risk of an infected cow 1/10,000
       Multiplied by
Amount of infectious material 1000 units
       Divided by
The number of vaccine doses 1,000,000
       Divided by
The reduction in infectivity related to the route of administration 200

This yields a risk estimate for vCJD of 1 case in 2 billion doses of vaccine [(1/10,000) x 1,000 x (1/1,000,000) x (1/200)]. A second scenario can also be considered, namely one in which a small amount of neural tissue inadvertently might contaminate the beef broth. We consider a 0.01% contamination with neural tissue. This would increase the amount of infectious material from 1000 units to 50,000 units, raising the total risk to 1 in 40 million. Because of the overestimates that were used in the risk calculation, the true risk is likely to be significantly less.

Potential sources of error

In estimating the risk of BSE contamination, it is important to note that each risk factor carries its own uncertainty. The overall risk, which is the product of these factors, compounds these uncertainties. For example, we have assumed no species barrier and no purification effect. The actual risk could be 10 to 1,000 fold lower, but probably no greater. On the other hand, we have assumed a 200-fold reduction due to an intramuscular route of administration. In fact, this risk could be 10-fold greater or 10-fold lower. Finally, in the case of viral vaccines, and based on experiments with analogous cell lines, we have assumed that BSE cannot replicate in cell cultures that were used. These uncertainties must be considered in order to correctly interpret the risk of BSE in viral vaccines. These calculations are not a formal risk assessment, but an attempt to estimate risk based on information currently available.

It should be noted that for both the viral and bacterial vaccine examples used, the exposure to this risk is temporary. Manufacturing changes have already been implemented which eliminate exposure during vaccine manufacture to bovine materials from countries at risk of BSE contamination. Vaccines made by these procedures are expected to be available in 2001.

Table 1

Estimated infectivity of bovine tissue by category
     
Category Tissue ID50/gram*
I Nervous tissue 107
II Spleen, lymph nodes, colon <2.5 x 104
III Pancreas, liver, lung <100
IV Muscle, bone, heart <0.1
     
Adapted from: Bader et. al, 1998 BioPharm *ID50/gram = number of infectious units per gram of tissue

References:

  1. Bader F, Davis G, Dinowitz M, Garfinkle B, Harvey J, Kozak R, Lubiniecki A, Rubino M, Schubert D, Wiebe M, and Woollett G, Assessment of risk of bovine spongiform encephalopathy in pharmaceutical products, Biopharm. Jan., 1998. pp. 20-31.
  2. Taylor DM, Fraser H, McConnell I, Brown DA, Brown KL, Lamza KA, and Smith GRA, Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie, Arch Virol 139:313-326, 1994.
  3. Bradley R, BSE Transmission studies with particular reference to blood, Dev. Biol Stand, 99:35-40, 1999.
  4. Kimberlin RH, An overview of bovine spongiform encephalopathy Dev Biol Stand 75:75-82, 1991.
  5. Donnelly CA, Ghani AC, Ferguson, NM, and Anderson RM, Recent trends in the BSE epidemic, Nature 389:903, 1997.
  6. Linda Detwiler, USDA
  7. Wilesmith JW, Wells GAH, Ryan JBM, Gavier-Widen D, and Simmons MM, A cohort study to examine maternally-associated risk factors for bovine spongiform encephalopathy, The Vet Record 141:239-243, 1997.
  8. Transcript of June, 2000 meeting of the FDA TSE Advisory Committee.
  9. Brown P, Cervenakova L, McShane LM, Barber P, Rubenstein R, and Drohan WN, Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt-Jakob disease in humans, Transfusion 39:1169-1178, 1999.
  10. Brown, P, Rohwer RG, Dunstan BC, MacAuley C, Gajdusek DC, and Drohan WN, The distribution of infectivity in blood components and plasma derivatives in experimental models of transmissible spongiform encephalopathy, Transfusion 38: 810-816, 1998..
  11. Harris, DA, Cellular biology of prion diseases, Clin. Mocro. Rev, 12: 429-444, 1999.

Table of Contents

 
Updated: March 20, 2001