3 new PNAS articles on prion seed capping and strain interference
Overproduction of Sup45 inhibits Sup35 seed formation
SAA fibril formations accelerated by injected seed
Strain interference in CJD
A model of prion capping in ARR sheep
Nature 392, 23-24 (1998) 5 Mar 98 Nature William J. Welch and Pierluigi Gambetti
"Prion diseases and neurodegenerative conditions such as Alzheimer's and Parkinson's have much in common - both seem to involve proteins which, when destabilized by (for example) genetic mutations, change conformation to form insoluble, pathogenic aggregates. Two papers now ask whether molecular chaperones might be involved in these folding changes, and they show that heat-shock proteins and prions can, indeed, interact in vitro."
3 articles in the 3 Mar 98 PNAS -- webmaster commentaryThere are three new articles in the 3 Mar 98 PNAS that more or less speak to the capping and strain interference issues and auxillarty factors.
1. The first article, highlights below, by Westermark et al. addresses SAA, a classical congophilic amyloidosis on Glenner's well-known list. (It has no relation to Alzheimer amyloid protein other than here too an amino terminal proteolytic fragment forms fibers.)
SAA is another 'infectious' and 'transmittable' protein-only disease, except without known practical significance and lacking viral enthusiasts. Here they show that preformed fibrils made from synthetic peptides and given in small doses i.v. in mice seed development of AA amyloidosis. By double immunogold labeling, the amyloid foci not only contained the synthetic fibrils but also murine protein AA fibrils. Mouse and mink each have several SAA isoforms, varying strongly in amyloidogenicity.
They observe further, "Formation of amyloid fibrils is supposed to occur as an off-pathway event from specific near-native protein intermediates at the folding-unfolding pathway. A nucleation mechanism is believed to be of importance because seeding a solution of amyloid fibril proteins like -protein or islet amyloid polypeptide with preformed fibrils made from the homologous proteins strongly enhances the speed by which new fibrils are formed. This fibril growth follows first-order kinetics."
2. The second article, highlights below, from the Susan Leibman lab at Chicago, concerns the yeast prion-like system psi. Yeast genetics allows far more sophisticated and faster experiments than in mammals. After getting past burdensome yeast terminological conventions, it seems that the binding of their two translation releasing factors causes excess Sup45 to inhibit seed formation of Sup35. These factors normally bind each other; excess sequesters Sup35, 'thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation.'
They still have not pinned down what an auxillary conformational factor called Pin+ does in facilitating conversion, it may be a more distal domain of Sup35 or another prion-like protein altogether. Excess Sup45p inhibits seed formation but has no effect on the propagation and does not alter the Pin status.
Factors like Pin and chaperones aren't essential as in concentrated solutions, Sup35 or its N-terminal fragment form de novo fibers even in the absence of other yeast proteins or Sup35 seeds, but probably still are important in vivo. The authors propose two classes of factors: general [chaparones like hsp 104 and proteins that degrade abnormal proteins] and specific factors (such as Sup45) that affect the genesis and propagation only of a single prion.
Conformational strain types were documented in Sup35 earlier. Extracts from PSI+ yeast strains also stimulate Sup35p conversion into an aggregated proteinase K-resistant form in psi extracts and induce fiber formation by Sup35p.
They note similarities to Alzheimer disease where 'apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid,' citing Evans, K. C et al (1995) PNAS 92, 763-767.
3. The third article, highlights below, is by Laura Manuelidis at Yale. As with the yeast article, there is some terminology to get through, here immunological metaphor instead of cryptic acronyms, such as vaccination [intracerebral injection], attenuated virus [longer incubation time] and expression of virulent agent [shorter incubation strain].
The strains used were a P102L from Japan propagated through rats before mice and a US case of sporadic CJD propagated through unsequenced guinea pig before mice.
There is a prior history reviewed somewhat here of multiple simultaneous strains in several species such as sheep and mink and also work at UCSF with transgenic mice that appeared after this article was submitted. Multiple strains is presumbably commonplace in vivo for populations like humans where heterozygotes are numerous. Various effects have been observed such as negative dominance and interference.
Interference is observed in this substantial study. This would seem to fit protein-only theory and capping of seeds, but another perspective [see full text of highlights below] is capably presented by the author.
PNAS Vol. 95, Issue 5, 2400-2405, March 3, 1998 Irina L. Derkatch, Michael E. Bradley, and Susan W. Liebman[PSI+], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI+]. However, another non-Mendelian determinant, [PIN+], is required for this induction.
We now show that SUP45 overexpression inhibits the induction of [PSI+] by Sup35p overproduction in [PIN+] strains, but has no effect on the propagation of [PSI+] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI+] strains.
We argue that excess Sup45p inhibits [PSI+] seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation.
This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid. Medline: Evans, K. C et al (1995) PNAS 92, 763-767
...Also, recent evidence suggests the existence of a non-Mendelian element, [PIN+], which is responsible for the ability of yeast [psi] strains to be induced to the [PSI+] state by Sup35p overproduction. [PIN+] can be eliminated by incubation on media containing guanidine hydrochloride (GuHCl) or by transient HSP104 inactivation (55). Unlike [PSI+], [PIN+] is not located in the N-terminal region of Sup35p and may either be a self-propagating determinant in the C-proximal part of Sup35p, or a prion domain in another protein that facilitates Sup35p conformational changes (55).
Although excess Sup45p inhibits [PSI+] seed formation, it has no effect on the propagation of [PSI+] and does not alter the [PIN] status of the cells.
Prions are generally viewed as self-propagating conformational protein variants that interact with and direct other molecules with the same amino acid sequence to fold into their prion conformation. Recent experimental data strongly supporting this view include the demonstration of in vitro and in situ conversion of PrPC into a proteinase K-resistant conformation in the presence of PrPSc (70 , 71) and the in vitro demonstration that protein extracts from [PSI+] yeast strains stimulate Sup35p conversion into an aggregated proteinase K-resistant form in [psi] extracts and induce fiber formation by purified Sup35p (41, 42).
...In concentrated solutions, Sup35p or its N-terminal fragment form fibers de novo even in the absence of other yeast proteins or Sup35pPsi+ seeds (42, 45). However, although these in vitro reactions appear to reflect the in vivo genesis and propagation of prions, it remains likely that both the genesis and propagation of prions in vivo are strongly influenced by other cellular factors.
One can imagine two classes of these factors. General factors, such as molecular chaperones or proteins that degrade abnormal proteins, are likely to interfere with the appearance and maintenance of a wide range of self-propagating protein conformations. One such factor, essential for the maintenance of both [PSI+] and [PIN+] (40, 54, 55), is the heat shock protein Hsp104, which is known to promote resolubilization of protein aggregates following heat shock (53).
Specific factors, which interact with either normal or prion conformational variants of a particular prion protein, are likely to affect the genesis and propagation only of a single prion. We argue that Sup45p, a protein known to complex with Sup35p (33, 34), is such a specific factor for [PSI+], because our data indicate that the overexpression of SUP45 interferes with the de novo induction of [PSI+].
The major observation is that transformation with high-copy SUP45 -containing plasmids causes a considerable reduction in the ability of transiently overproduced Sup35p to induce the appearance of Ade+ colonies, indicative of [PSI+]. Although this is likely to be because of a negative effect of excess Sup45p on [PSI+] induction, it could be also explained by a Sup45p-stimulated reduction in the [PSI+] suppression phenotype or by an incompatibility of [PSI+] and excess Sup45p.
We eliminate the latter possibilities by showing that SUP45 overexpression does not inhibit growth of [PSI+] cells with normal or elevated Sup35p levels, does not reduce the efficiency of readthrough of an ade1-14 nonsense allele, and does not cause the elimination of [PSI+] .
In contrast, SUP45 overexpression rescues the inhibition of growth caused by Sup35p overproduction in [PSI+] derivatives and increases the nonsense suppression in derivatives containing weak [PSI+] variants. These phenotypes are likely to be typical of major inducible [PSI+] types, because in our experiments we used different [PSI+] variants induced by Sup35p overproduction.
Furthermore, overexpression of SUP45 in unrelated [PSI+] strains (where [PSI+] was not induced by Sup35p overproduction) also caused an increase in suppression efficiency (59), appeared to be compatible with [PSI+] (59, 73), and allowed for SUP35 overexpression (33). Thus, we are left with the conclusion that SUP45 overexpression reduces the efficiency with which Sup35p overproduction can induce [PSI+].
Our finding that excess Sup45p rescues the lethality of excess Sup35p in [PSI+] strains can be explained by the model proposed by Paushkin et al. (34) that Sup35p overproduction causes growth inhibition because too much Sup45p is sequestered in [PSI+] aggregates. However, because Sup45p is reported to be in [PSI+] aggregates in some (34) but not the other (40) studies, this model is unproven.
... The fact that Sup45p overexpression in [PSI+] strains has an allosuppressor rather than an antisuppressor effect is not consistent with the Sup45p sequestration hypothesis (34). We attribute [PSI+] -associated suppression to the lack of functional Sup35p and propose that SUP45 overexpression might increase the level of translational readthrough by further unbalancing the translational termination machinery. Because Sup45p overdose inhibits [PSI+] induction but not [PSI+] propagation or stability, it must uniquely affect the step of [PSI+] seed formation.
The same step is apparently affected by [PIN+], another factor involved in [PSI+] biogenesis. [PIN+] is a non-Mendelian element that determines whether [PSI+] can be induced de novo by the overproduction of Sup35p (55). The molecular basis of the [PIN+] factor is unknown and could involve a prion form of a general molecular chaperone, a prion protein that exclusively affects Sup35p conformational liability, or a new Sup35p prion variant distinct from [PSI+] and determined by the conformation of a region in the C-proximal part of Sup35p (55).
Transient SUP45 overexpression did not cause any detectable loss or induction of [PIN+]. Thus, excess Sup45p and Sup45p/Sup35p binding are unlikely to induce a permanent conformational change in either Sup35p or Sup45p affecting the [PIN] status of the cell. Furthermore, the [PIN+] determinant is unlikely to be a prion form of Sup45p, because in that case an excess of Sup45p would be expected ( 12) to induce [PIN+]. The finding that SUP45 overexpression does not inhibit the propagation of existing [PSI+] is consistent with the report (34) that Sup35p domains capable of binding Sup45p are not located in the N-terminal [PSI+] domain, because this means that Sup35p bound to Sup45p is presumably still able to join existing [PSI+] aggregates via the N-terminal domain.
In contrast, de novo formation of [PSI+] seeds may require a rare spontaneous Sup35p conformational switch or Sup35p/Sup35p intermolecular interactions. Either process would be more efficient when Sup35p is in excess. However, the situation apparently changes when the excess in Sup35p is balanced by an excess in Sup45p. If seed formation is preceded by a conformational change in a Sup35p molecule, Sup45p might inhibit this event by stabilizing the Sup35pPsi conformation. Alternatively, Sup45p binding may inhibit Sup35p from interacting with other soluble Sup35p molecules, thereby inhibiting seed formation.
PNAS 95, Issue 5, 2558-2563, March 3, 1998 Westermark et al.[Don't confuse SAA amyloid with Alzheimer amyloid -- these are diseases of different and unrelated genes -- webmaster]
Highlights of the article:
Secondary or amyloid protein A (AA) amyloidosis is a life-threatening systemic disease in which deposits of amyloid can be found in most tissues of the body. The main constituent of the amyloid deposits is a fibril formed by beta-pleated sheets of protein AA, the latter of which is an N-terminal 44- to 100- amino acid cleavage product of the 104 amino acid precursor serum AA (SAA) (for review, see ref. 1). [
In humans, there are at least three different SAA genes coding for SAA1, SAA2 and SAA4, respectively (2). SAA1 and SAA2 are acute phase reactant high density apolipoproteins. These are expressed mainly by the liver, but SAA4 is expressed in several different tissues (3, 4). N-terminal fragments of SAA1 and SAA2 give rise to protein AA and polymerize to amyloid fibrils in humans. SAA is a conserved protein, and AA amyloidosis occurs, with varying frequency, in many mammalian species and in some birds (for review, see refs. 5 and 6).
The mouse (Mus musculus) is prone to develop AA amyloidosis and is the most commonly used animal model for AA amyloidosis. The two mouse SAA isoforms of high density lipoprotein, SAA1 and SAA2, are both acute phase reactants, but only SAA2 is amyloidogenic and is found as protein AA in the fibrils (7, 8). The plasma concentration of SAA is normally low both in humans and in mice, but the SAA concentration rises quickly at an acute inflammation as a response to cytokines, especially interleukin-1, interleukin-6, and tumor necrosis factor (for review, see ref. 9), to levels as high as 1 mg/ml. When inflammation subsides, the SAA concentration gradually returns to normal.
In humans, AA amyloidosis most commonly occurs in individuals with long term active inflammatory disease, which, in developed countries, usually is rheumatoid arthritis. In developing countries, the common cause of AA amyloidosis is a chronic infectious disease among which tuberculosis, leprosy, and malaria predominate (10).
However, most individuals with these chronic inflammatory diseases never develop AA amyloidosis despite the high plasma concentration of SAA. It is still unclear why only a subset of individuals develops AA amyloidosis. In contrast to the disease in the mouse, there is so far no definite proof of a specific amyloid-prone SAA variant in humans, although some SAA isotypes may be over represented as amyloid fibrils (11, 12). Therefore, in addition to high concentrations of an amyloidogenic protein, other factors are important in the pathogenesis of AA amyloidosis.
AA amyloidosis can be easily induced experimentally in many strains of mice by a prolonged inflammatory challenge, e.g. s.c. injections of silver nitrate or casein (13). It also was observed several decades ago that an extract of mouse amyloid tissue, given i.v. with the simultaneous induction of inflammation, dramatically shortens the time for AA amyloid to develop (14). The same effect can be achieved by injection of cells from mice with AA amyloidosis (15).
Despite many efforts, the active component [called "amyloid enhancing factor" (AEF)] has never been isolated or defined. It has, however, been suggested to act like the infectious protein in scrapie-related cerebral diseases (16). Because a general consensus concerning how amyloid deposits develop includes the role of a nidus, we previously tested (17) the effect of synthetic amyloid-like fibrils made from short peptides on the time necessary for amyloid induction and found an AEF-like effect of these fibrils. We now present strong evidence that AEF-like activity is exerted by synthetic amyloid-like fibrils by serving as a nidus on which new AA amyloid fibrils form.
C-terminally amidated synthetic peptides used for in vitro formation of amyloid-like fibrils
YSNNFGAILSS CPLMVKVLDAV PAINVAVHVFRK YTIAALLSPYS SYSTTAVVTN
In this study, we show that preformed fibrils made from synthetic peptides and given in small doses i.v. in mice shorten the lag phase between an inflammatory stimulus and development of AA amyloidosis and, consequently, have amyloid-enhancing effects. By the use of radioactivity-labeled synthetic fibrils, we followed injected synthetic fibrils to the lung and spleen. Some fibril aggregates were trapped in lung capillaries and formed small distinctive amyloid foci that could be identified by Congo red staining. By double ImmunoGold labeling, we were able to show that these amyloid foci not only contained the synthetic fibrils but also murine protein AA fibrils.
Likewise, we found that early perifollicular splenic amyloid deposits contained radioactivity labeled material, indicating that synthetic fibrils not only were trapped in the lung but also were present at an area known to be a target for the first deposits in murine experimental AA amyloidosis (25). Sections of kidney and liver, organs in which amyloid deposits occur later (25), exhibited no radioactivity.
Formation of amyloid fibrils is supposed to occur as an off-pathway event from specific near-native protein intermediates at the folding-unfolding pathway (26). A nucleation mechanism is believed to be of importance because seeding a solution of amyloid fibril proteins like -protein (27) or islet amyloid polypeptide (28) with preformed fibrils made from the homologous proteins strongly enhances the speed by which new fibrils are formed. This fibril growth follows first-order kinetics (29, 30).
The nucleation mechanism is similar to that implicated in the prion model of "infectivity"; the protein with abnormal tertiary structure serves as template for other protein molecules (16, 31). However, in prion diseases, the infectivity may be dissociated from the formation of amyloid fibrils (32).
The formation of the nucleus in the amyloidogenesis in general is, however, poorly understood, but it should be noted that in the present experiments only peptides in their fibrillar form exerted amyloid-enhancing effects. The finding that amyloid-like fibrils synthetically made from heterologous proteins enhanced the development of AA amyloidosis presumably by acting as nidi is of great interest. This finding raises the provocative question of whether other components in addition to amyloid fibrils can act as nidi in the amyloidogenesis.
One of the major and unsolved questions in the pathogenesis of human AA amyloidosis asks why only some individuals with longstanding inflammatory conditions and high SAA plasma concentration develop amyloid deposits. It is known from the mouse (33) and the mink ( 1, 34) that several SAA isoforms exist and that they vary strongly in amyloidogenicity.
Such a mechanism may exist also in humans (11, 12) but is probably less important because in most instances, human AA amyloid is derived from common SAA isotypes also present in individuals who do not develop amyloidosis. Therefore, other factors in addition to a suitable SAA must be of importance in the human amyloidogenesis. The mechanism found in the present study in which exogenous substances enhance AA-amyloidogenesis in an experimental mouse model may be true also in the human situation. Hypothetically, an exogenous substance may form a nidus on which the first AA fibrils form. Such a mechanism could explain why only a fraction of individuals with longstanding high plasma SAA concentration get AA amyloidosis. Furthermore, similar mechanisms may be important not only in AA amyloidosis but also in other types of amyloid deposits.
PNAS 95, Issue 5, 2520-2525, March 3, 1998 Laura ManuelidisAlthough slow and persistent viruses often escape host defenses infection may be prevented by live vaccines. To determine whether an attenuated "slow" strain of the Creutzfeldt-Jakob disease agent (SY) could block expression of a virulent "fast" strain (FU), outbred CD-1 mice were inoculated intracerebrally with low infectious doses of SY and challenged 80 days later with higher doses of FU.
For comparison, the same SY and FU samples were inoculated in two parallel control groups. All 18 superinfected mice showed incubation times identical to those inoculated with only the SY strain, yielding clinical disease >110 days later than predicted for the FU strain. Neurological signs, such as scratching and an extended clinical phase, were also characteristic for SY but not FU infection. Moreover, the widespread cortical pathology of FU was not detectable in superinfected mice.
Western blot analyses further showed no strain-specific differences in prion protein (PrP) band profiles for all experimental groups, although there was ~10-fold more protease-resistant PrP (PrP-res) in FU brains during terminal disease. In contrast, infectivity assays revealed an ~10,000-fold difference between SY and FU at terminal stages, indicating that PrP-res content does not correlate with infectivity.
In summary, an attenuated strain of the Creutzfeldt-Jakob disease agent evokes substantial interference against a virulent agent. Because superinfected mice had little PrP-res just before the onset of clinical disease and retained abundant cellular PrP, cellular PrP was not the factor limiting FU replication. The mechanisms underlying SY interference are not understood but could be based on host recognition of foreign molecular features shared by this class of invasive agents involving antibody production, and possibly involve defective viral particles produced by attenuated variants.
The introduction of live viruses to control infection was practiced by the Chinese for centuries and was brought from Constantinople by Lady Montague before Jenner's work was published in 1798 (1). The vaccination strategy derived from the fundamental observation that less-fulminant infectious lesions contained attenuated agents able to prevent superinfection by more virulent ones. By the 1960s, attenuated mutant strains were selected by sequential passage in animals and cell cultures, leading to the live poliovirus vaccine (2).
Today, genetic engineering provides the potential to design and manufacture more attenuated viral vaccines that lack pathogenicity. The effectiveness of many vaccines is based on their ability to provoke specific antiviral antibodies in the host. However, some slow and persistent viral infections such as HIV are not arrested by these host responses, and persistent and latent viruses exploit a variety of mechanisms to evade immune surveillance (3).
The persistent infectious agents causing the neurologically devastating and incurable Creutzfeldt-Jakob disease (CJD) of humans, as well as sheep scrapie and bovine encephalopathy (BSE), have an intracellular residence and escape typical immunological recognition. Hence, an invasive form of these agents may be required to achieve effective vaccination. Presumably mechanisms for strain interference, if observed, would depend on host recognition of the foreign agent or on the positioning of the attenuated agent on sites required for the life cycle of the virulent challenge agent.
The latter concept was suggested in an original scrapie study 25 years ago (4). Since that time, interference effects have not been reported with any combination of strains other than scrapie 22A and 22C. The two other previous experiments were also complicated by host genetic factors because there was a reversal in the virulence of both agent strains in congenic VM mice as compared with other inbred mice. Moreover, incubation time was also the only data shown to support the inteference effect (5, 6), and host prion protein (PrP) was not analyzed. Host PrP has been postulated to convert into an infectious or "prion" form (7), although this idea remains controversial.
Interference might also be based on intracellular mechanisms that antecede the evolutionary development of more sophisticated immune responses. Such mechanisms may include lysosomal compartmentalization ( 31) or a variety of chemokines and intracellular products. A nonspecific response to injected homogenates is excluded because interference effects were not reproduced by previous inoculation with noninfectious brain homogenates. Additionally, interference required administration of the slow scrapie strain at least 35 days before superinfection (4), an observation that implicates host recognition of the foreign agent and, possibly, agent processing or complementation by an endogenous virus (36 , 37). Finally, host recognition may be responsible for significant agent clearance, making the doubling time of attenuated agents appear deceptively long and obscuring a faster and more conventional viral replication time.
The most parsimonious explanation for interference is the assumption that these agents contain a viral genome that specifies strains. Indeed, when one reviews all the reproducible data in this field, there are no biological, physical, or molecular data that exclude a viral agent (14, 21, 33). Because an invading infectious agent prevents superinfection, the attenuated agent itself, or the response that it elicits, must be responsible for interference. The attenuated strain SY may produce many defective interfering particles (without core nucleic acid) or viral products that are capable of blocking the cellular sites needed for completion of its own life cycle. Such products, or the defective particle itself, could inhibit the second challenge agent. This type of mechanism is well described for some viruses. In this case significant production of viral protein(s) that would not partition with infectious particles during fractionation and the accumulation of specific viral protein(s) at early stages of infection would be expected. An alternative but not mutually exclusive mechanism for interference centers on host recognition of one or more foreign features common to both strains. This host response could involve cryptic inflammatory, immune, or intracellular pathways. Experiments with prolonged rat CJD infections indicate that host inflammatory responses to infection can occur well before the onset of clinical disease (32) and lymphocytes can infiltrate the brain early in scrapie (34). Indeed, attenuated agents that produce prolonged disease may be those that are less capable of escaping typical host immune mechanisms, and a number of factors could modulate host antibody effects to some strains (33). There have been very few antibody studies in scrapie and CJD, and most of these were done many years ago (e.g., ref. 35). All investigators have used very crude preparations for inoculation and similarly crude material (with many proteins and very small amounts of agent) to monitor specific immunoglobins. Given the current data, a reevaluation of agent-specific antibodies seems reasonable.
The most parsimonious explanation for interference is the assumption that these agents contain a viral genome that specifies strains. Indeed, when one reviews all the reproducible data in this field, there are no biological, physical, or molecular data that exclude a viral agent (14, 21, 33). Because an invading infectious agent prevents superinfection, the attenuated agent itself, or the response that it elicits, must be responsible for interference.
The attenuated strain SY may produce many defective interfering particles (without core nucleic acid) or viral products that are capable of blocking the cellular sites needed for completion of its own life cycle. Such products, or the defective particle itself, could inhibit the second challenge agent. This type of mechanism is well described for some viruses. In this case significant production of viral protein(s) that would not partition with infectious particles during fractionation and the accumulation of specific viral protein(s) at early stages of infection would be expected.
An alternative but not mutually exclusive mechanism for interference centers on host recognition of one or more foreign features common to both strains. This host response could involve cryptic inflammatory, immune, or intracellular pathways. Experiments with prolonged rat CJD infections indicate that host inflammatory responses to infection can occur well before the onset of clinical disease (32) and lymphocytes can infiltrate the brain early in scrapie (34). Indeed, attenuated agents that produce prolonged disease may be those that are less capable of escaping typical host immune mechanisms, and a number of factors could modulate host antibody effects to some strains (33).
There have been very few antibody studies in scrapie and CJD, and most of these were done many years ago (e.g., ref. 35). All investigators have used very crude preparations for inoculation and similarly crude material (with many proteins and very small amounts of agent) to monitor specific immunoglobins. Given the current data, a reevaluation of agent-specific antibodies seems reasonable.
... Further experiments using several peripheral routes of infection including oral administration and inoculation of lower doses of attenuated strains should be informative. There are also established laboratory strains that when passaged in a different species show little pathogenicity for their host of origin.
Because low doses of the mouse-passaged SY agent showed a strong blocking effect, an agent selected in a different species might be sufficient to supress infection in the original host from which it derived. Thus infectious agents causing negligible disease or very prolonged incubation in mice, such as the 263K hamster-passaged scrapie strain, may prevent superinfection by more virulent mouse-passaged agents, including those isolated from BSE. Similarly, several CJD agents that have been propagated and selected in this laboratory may block more virulent strains of CJD in primates if they retain sufficient invasiveness to be recognized by the host but have little or no pathogenicity.
In contrast, a killed vaccine may not retain this effect. Indeed, only treatment of a "slow" scrapie strain with 5 Mrads of ionizing radiation or 12 M urea completely abrogated an interference effect, whereas other treatments did not (6). However, partial denaturation of reasonably purified infectious preparations may further vaccine development if such treatments expose additional agent components that are correctly delivered to and recognized by the host.
Chemical modifications of purified infectious preparations may also generate more completely attenuated structures that can propagate and preserve their interfering properties, and clearly the elucidation of intrinsic agent molecules should further the development of recombinant vaccines. The present results suggest reasons for a renewed interest in a live vaccination approach to prevent a spectrum of slow viral infections including HIV. Nevertheless, it is important to emphasize that with CJD and other agents of this class, it is premature to undertake any experiments outside a controlled laboratory setting without strong evidence showing an attenuated agent is incapable of causing late-onset neurodegenerative disease.
3 Mar 98 Alex Bossers Institute for Animal Science and Health
|Question: If the ARR-PrPc caps and blocks the
growing PrPSc polymer, this would stop the reaction. Wouldn't this
completely inhibit the conversion in vitro? I would think that a low
efficiency of the conversion reaction would explain your in vitro
result well. if the resistance is so complete, then ARR should immunize them.
Response: Basically this is right and could be happening in vivo. A low conversion efficiency of the ARR variant could declare the observed results. However, it is hard to imagine the absolute resitance of ARR/ARR sheep by i.c. inoculation with different scrapie/BSE isolates. If there would be a low conversion rate than at least at some point you would expect to see some sort of neuropathological effects or PRP-Sc accumulation in experimentially infected ARR/ARR sheep.
That's why I postulated the second hypothesis of capping the PrP-Sc polymer. In more detail: the PrP-Sc seed (or agent?) 'infects' a ARR/ARR sheep. The ARR PrP-c molecules bind to the nucleating site of the PrP-Sc seed by which the ARR molecule changes in conformation giving it protease resistance but also rendering the new resulting nucleation site (secondary) inactive or at least this new nucleation site is changed in such a manner that new ARR PrP-c moldecules are unable to bind.
For ARR protein to be therapeutic, several points need to be addressed: