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Below is a letter sent to Gavin Thomson on the subject of FMD and oral interferon.


Dear Dr. Thomson,

Your excellent article entitled Foot and mouth disease in wildlife in: Virus res 91 (2003) 145-161 was just reviewed.
I wish to acquaint you with the literature on FMD and interferon alpha and to propose research collaboration. If you are interested, please let me know.
In the past I worked with Dr. Alan Young in Nairobi on East Coast Fever.

Data show that it is not interferon alpha (IFNa) in the blood, but instead it is interferon stimulated genes (ISG) which perform the immune modulation we seek in animal health.  ARS scientists have shown that blood levels of IFNa are not the hallmark of protection against FMD.  In December 2003, ARS scientists reported that Ad5-pIFNa injected 1, 3 or 5 days before challenge with FMD virus resulted in complete protection.1 
On page 277 of this article, in the discussion the authors stated, "It was interesting to note that animals challenged 7 days after Ad5-pIFNa administration had no detectable IFNa in their plasma for 2-3 days prior to challenge; yet they had delayed and lower levels of viremia and vesicular lesions as compared to the control animals. This data implies that although complete resistance to virus replication may only last 1-2 days after clearance of IFNa , IFN "activated" cells can still reduce the rate of virus replication for an additional 1-2 days. I believe that it is reasonable to conclude that ISG induced by IFNa administration provided the protection which lasted longer than the IFNa .

There are more than 600 genes modulated by interferon (see www.lerner.ccf.org/labs/williams). Gene expression analysis using specific DNA fragments aligned onto microchips (microarrays) uncover the biological basis of diseases, enhance our understanding of host-pathogen interactions and provide rational avenues for immunotherapy.2-5 The microarrays will identify which genes are activated in cells by detecting the presence or absence of a corresponding messenger RNA (mRNA). When stimulated, cells convert genetic information into proteins, by first forming the corresponding
intermediate product, the mRNAs. Therefore, if the mRNA correspondingto a gene is found in cells, it is likely that the specific protein is also produced by cells.4

In addition to 2'5' oligoadenylate synthetase (2'5' AS),6 orally administered IFNa has been shown to affect systemic phenotypic expression of lymphocyte populations. IFN activated NK1.1, CD11b+, CD19/CD20 B-cells, and subpopulations of T-cells are present in the peripheral circulation of tumor bearing mice as early as 4 hours after the initiation of oromucosal IFNa therapy. In addition, oromucosal IFN therapy also induced trafficking of cells from both the spleen and
peripheral lymph nodes to the site of tumor cell replication.
Other genes upregulated by oral IFNa include Crg2 and other chemokines, proteases associated with antigen processing, and genes involved in lymphocyte activation, apoptosis, and protein degradation.7
As shown by scientists at ARS, the FMD virus possesses the ability to overcome several key host defenses.
FMD virus inhibits production of IFNa /b 8 and blocks a key IFN-inducible, antiviral pathway, i.e.- double-stranded RNA (dsRNA)-dependent protein kinase R (PKR).9 The other key IFN-inducible, antiviral pathways are the Mx proteins (Mx) of the Dynamin super family and the 2'5' AS that activates ribonuclease RNaseL.10

Some ISG are up-regulated within a few hours after oral IFN administration.11,12 A 15kDa protein called ISG-15 is up-regulated in human buccal epithelial cells in vivo and in vitro with a peak level of ISG-15 detected 2 hours after oral HuIFNa administration.11 Up-regulation of Mx proteins was detected in the spleen of mice, and in the peripheral blood mononuclear cells of humans, 2-4 hours after murine IFNa or HuIFNa , respectively, were ingested.12 These data demonstrate that orally administered IFNa has rapid and systemic positive biological effects in animals and humans.

In protection experiments, cattle given coital vesicular exanthema virus and then infected with FMD virus developed a milder form of FMD and developed FMD later than control calves infected with FMD alone.13 A protective effect was noted in cattle infected with FMD virus if the cattle were given multiple injections of yeast RNA.14 Presumably, the induction of IFN by virus and RNA, respectively, resulted in ISG expression responsible for the protection noted in these studies.13,14

Low levels of circulating IFNa induced by infectious bovine rhinotracheitis (IBR) virus have been detected in the sera of
calves,15,16 and IFNa or IFNa -like viral inhibitors appear in the nasal secretions (NS) of cattle after intranasal administration of IBR virus.17-21 Straub and Ahl reported that IFN induced in the NS by IBR virus given intranasally provided protection against FMD viral challenge.18 One or 2 days after intranasal vaccination with IBR virus, calves were challenged with FMD virus. Interferon was detected in the NS within 24 hours of vaccination, and IFN persisted at high levels in the NS (not the blood) for 6 additional days and at low levels through the tenth day after IBR virus inoculation. Vaccinated calves had a milder course of FMD and more than a 99% reduction in FMD virus titers in the NS.18

In the process of studying FMD virus transmission from carrier to susceptible cattle, carriers of FMD virus were inoculated intranasally with IBR virus in an effort to create a stress which might increase excretion of FMD virus from carrier cattle. However, FMD virus disappeared from the esophageal-pharyngeal fluid of the 2 carrier animals 1 day after IBR virus inoculation and was not detected again during the 4-week sampling period.22 Interferon was not assayed in this experiment, but it would have been induced by IBR virus for 5-7 days and the IFN would induce ISG which inhibited the FMD virus in
these carrier animals.
The report that FMD was not detected for 4 weeks exceeds (by 3 weeks) the time of IFNa production time of NS interferon from vaccination. 
Clearly, IFN is readily induced in the NS of feedlot calves by IBR viral vaccine;19,23 perhaps revaccination with intranasal IBR viral vaccines offers the cattle industry an inexpensive tool to help control FMD.

Induction of an Interferon Response with Synthetic Inducers.
The use of a viral inducer of IFN in cattle with FMD is in agreement with the successful use of oral synthetic IFN inducers which protected mice from a subsequent infection with FMD virus.24 Richmond and Campbell reported that one oral IFN inducer protected mice if given 2, 24 or 48 hours before FMD virus inoculation and another inducer protected mice if given 18 hours or less before FMD virus.24 A single injection into mice of 150 g of the synthetic IFN inducer polyriboinosinic:
polyribocytidylic acid (PolyI:C) 18 hours before a challenge with 100 LD50 of FMD virus (strain Asia-10) was 100% protective.25 However, PolyI:C is toxic when tested in cattle.26-28

Cunliffe et al 29 reported that PolyI:C (1 mg/kg weight) was given intraperitoneally (IP) to 5 pigs one day before the 5 treated pigs (and 3 untreated control pigs) were placed in a room with 2 pigs with FMD. The controls exhibited signs of FMD on 3, 4, and 4 (mean 3.7) days after FMD virus exposure whereas the PolyI:C-treated pigs exhibited signs of FMD on 4, 7, 7, 7 and 7 (mean 6.4) days after FMD virus exposure. All pigs given PolyI:C exhibited severe but transient adverse effects (pruritus, trembling, urination, defecation and ataxia). Despite the toxicity, treated pigs experienced a delay in development of FMD, compared to controls.

McVicar et al reported that various concentrations of PolyI:C were given intravenously (IV) to 11 calves (0.25 - 4 mg/kg weight) and 13 goats (1-4 mg/kg). Two calves given PolyI:C were tested hourly for 6 hours for serum IFN which was detected at each hour. All calves given PolyI:C had a transient temperature increase. Calves were challenged intramuscularly (IM) or intradermalingually (IDL) with 108 PFU of FMD virus 2-6 hours after PolyI:C was given. Differences were not noted in clinical FMD or viremia between treated and control calves.30

Goats given PolyI:C had a mean temperature increase of 2.3* F versus 0.5* F in controls given placebo. One goat died within 24 hours and one goat aborted within 48 hours of PolyI:C treatment. Most goats given PolyI:C showed signs of toxicity. Twelve goats given PolyI:C and 7 controls were challenged IN with 104 PFU of FMD virus. Differences were not noted in clinical FMD, viremia or pharyngeal carriers during the 14 days after challenge.30  It is reasonable to conclude that
toxic IFN inducers are not a useful tool in the control of FMD.

Oral Administration of Interferon to Cattle.
The oral delivery of  HuIFNa has been beneficial to cattle undergoing shipping fever, or challenged with virulent IBR virus or Theileria parva.31-34 In studies involving 7,000 feeder cattle, a single dose of orally administered human HuIFNa at the time of diagnosis of respiratory disease, given with antibiotics, reduced mortality significantly (p<0.001), when compared to feeder calves given placebo and antibiotics.31 The clinical effects of a virulent IBR virus challenge in feeder calves were significantly reduced by oral HuIFNa therapy.32 In studies of naturally occurring shipping fever, oral HuIFNa given for 3 days before shipping, or once after arrival, improved weight gain or reduced illness.33 In a challenge study of calves given Theileria parva, the causative agent of East Coast Fever, some calves given oral HuIFNa survived an otherwise fatal challenge.34 In the 4
 studies cited above,31-34 the beneficial oral dose of HuIFNa was less than 500 international units (IU) per calf.  Blood levels of IFNa are not detectable after administration of 500 IU of HuIFNa, yet immune modulation is evident.

Novel FMD Viral Disease Control Strategy. In January 2003, scientists at ARS on Plum Island reported that a recombinant
replication-defective human adenovirus type 5 vector containing porcine IFNa (Ad5-pIFNa ) was constructed.35 When the Ad5-pIFNa was injected into swine, the resulting IFNa production completely protected 3 swine given virulent FMD virus. Swine with IFNa showed no signs of FMD, did not develop viremia and did not develop antibodies against viral nonstructural proteins. However, when Ad5-pIFNa or this same vector modified to carry bovine IFNa was injected into cattle, only Ad5-pIFNa provided partial in vivo protection by delaying viremia 1 day and decreasing vesicle formation..36 The studies are further proof of the benefit of IFNa (and ISG) in the control of FMD.  The limiting factor in the use of this injectable vector is the need for injections.  An interferon delivered in food or water would be preferred.

Mechanism of Interferon-Induced Antiviral Efforts. The 3 intracellular IFN-induced antiviral pathways are the Mx, PKR and the 2'5' AS pathways.10 The PKR is a ubiquitously expressed serine/threonine protein kinase induced by IFN and activated by dsRNA, cytokines, growth factors and stress signals.6,10,37 The antiviral activity of PKR is mediated by its phosphorylation of the a subunit of initiation factor eIF2. Phosphorylation of eIF2 prevents recycling of eIF2:GDP2 to eIF2:GTP, trapping the recycling factor eIFa and resulting in rapid inhibition of translation.6,10 PKR also mediates programmed cell death (apoptosis) induced by dsRNA.10,37,38 Both inhibition of protein synthesis and induction of apoptosis restrict viral replication and spread.10 However, FMD virus has the ability to suppress IFNa /b production.8 It has recently been reported by scientists at ARS that PKR has an important role in the inhibition of FMD virus replication.9
A variant of FMD virus lacking the coding region for the leader (L) proteinase was developed, and this tool resulted in the identification of the role of PKR inhibition by FMD virus.9,39 Since a FMD viral strategy for control of host cells is to: 1) suppress IFNa /b production, and 2) block the effect of PKR, then treatment with exogenous IFNa will supplement endogenous IFNa production in the event of an FMD viral challenge.

REFERENCES

1. Moraes MP, Chinsangaram J, Brum MCS, Grubman M.
Immediate protection of swine from foot-and-mouth disease: a combination of adenoviruses expressing interferon alpha and a foot-and-mouth disease virus subunit vaccine. Vaccine 22: 268-279, 2003.

2. Der SD, Zhou A, et al. Identification of genes differentially regulated by interferon ", $, or ( using oligonucleotide arrays. Proc Natl Acad Sci U S A 95(26):15623-16528, 1998.

3. Ren B, Robert F, et al. Genome-wide location and function of DNA binding proteins. Science 290:2306-2309, 2000.

4. Dhiman N, Bonilla R, et al. Gene expression microarrays: a 21st century tool for direct vaccine design. Vaccine 20:22-30, 2001.

5. Huang Q, Liu D, et al. The plasticity of dendritic cell responses to pathogens and their components. Science 294:870-875, 2001.

6. Williams, B.R.G. (2000). Interferons. In: Encyclopedia of Microbiology, Volume 2. 2nd ed. J. Lederberg (ed.) New York: Academic Press, pp. 826-841.

7. Tovey, M., Lallemand, C., Meritet, J-F., Maury, C. (2003). Oromucosal interferon therapy: mechanism(s) of action. Presentation at annual meeting 2003 ISICR, Cairns, Australia.

8. Chinsangaram J, Piccone ME, et al. Ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon. J Virol 73(12):9891-9898, 1999.

9. Chinsangaram J, Koster M, et al. Inhibition of L-depleted foot-and-mouth disease virus replication by alpha/beta interferon involved double stranded RNA-dependent protein kinase. J Virol 75(12): 5498-5503, 2001.
 
10. Williams BRG. Interferons. In: Encyclopedia Micro. Vol. 2, 2nd ed. Academic Press. pp. 826-841, 2000.

11. Smith JK, Siddiqui AA, et al. Oral use of interferon-" stimulates ISG-15 transcription and production by human buccal epithelial cells. J Interferon Cytokine Res 19(8):923-928, 1999.

12. Brod SA, Nelson L, et al. Ingested interferon alpha induces Mx RNA. Cytokine 11(7):492-499, 1999.

13. Kubin G. Interferenz zwischen dem virus des blaschenausschlages des rindes und dem virus der maul- und klauenseuche.
Wien Tierarztl Monatsschr 48:265-277, 1961.

14. Thely M, Choay J, et al. Virostasis induced in vivo by non-infectious ribonucleic acids. C R Acad Sci (Paris) 256:1048-1050, 1963.

15. Rosenquist BD, Loan RW. Interferon induction in the bovine species by infectious bovine rhinotracheitis virus. Am J Vet Res 30(8):1305-1312, 1969.

16. Cummins JM, Rosenquist BD. Effect of hydrocortisone on the interferon response of calves infected with infectious bovine rhinotracheitis virus. Am J Vet Res 38(8):1163-1166, 1977.

17. Ahl R, Straub OC. Local interferon-production in the respiratory-and genital tract following infection with rhinotracheitis (IBR) and herpes exanthema (IPV) virus. Dtsch Tierarztl Wschr 78(24):653-655, 1971.

18. Straub OC, Ahl R. Lokale interferonbildung beim rind nach intranasaler infektion mit avirulentem IBR/IPV-virus und deren wirkung auf eine anschlieffende infektion mit maul- und klauenseuche-virus. Zbl Vet Med 23:470-482, 1976.

19. Todd JD, Volenec FJ, et al. Interferon in nasal secretions and sera of calves after intranasal administration of a virulent
infectious bovine rhinotracheitis virus: Association interferon in nasal secretions with early resistance to challenge with virulent
virus. Infect Immun 5(5):699-706, 1972.

20. MacLachlan NJ, Rosenquist BD. Duration of protection of calves against rhinovirus challenge exposure by infectious bovine rhinotracheitis virus-induced interferon in nasal secretions. Am J Vet Res 43(2):289-293, 1982.

21. Cummins JM, Rosenquist BD. Partial protection of calves against parainfluenza-3 virus infection by nasal-secretion interferon induced by infectious bovine rhinotracheitis virus. Am J Vet Res 43(8):1334-1338, 1982.

22. McVicar JW, McKercher PD, et al. The influence of infectious bovine rhinotracheitis virus in the foot-and-mouth disease carrier state. Proc 80th Ann Meeting US Anim Health Assoc 254-261, 1974.

23. Cummins JM, Hutcheson DP. Effect of interferon on feedlot cattle. Bovine Proc 15:109-115, 1983.

24. Richmond DY, Campbell CH. Foot-and-mouth disease virus: Protection induced in mice by two orally administered interferon inducers. Arch Ges Virusforsch 42(1):102-105, 1973.

25. Richmond JY, Hamilton LD. Foot-and-mouth disease virus inhibition induced in mice by synthetic double-stranded RNA
(polyriboinosinic and polyribocytidylic acids). Proc Nat Acad Sci U S A 64(1):81-86, 1969.

26. Rosenquist BD. Polyriboinosinic-polyribocytidylic acid-induced interferons in calves. Am J Vet Res 32(1):35-39, 1971.

27. Theil KW, Mohanty SB, et al. Effect of PolyI:C on infectious bovine rhinotracheitis virus infection in calves. Proc Soc Exptl Biol Med 137:1176-1179, 1971.

28. Angulo AB, Savan M. Preliminary studies in interferon induction on the respiratory tract of cattle. Proc 74th Ann Meeting US Animal Health Assoc 577-583, 1970.

29. Cunliffe HR, Richmond JY, et al. Interferon inducers and foot-and-mouth disease vaccines: influence of two synthetic
polynucleotides on antibody response and immunity in guinea pigs and swine. Can. J Comp Med 41:117-121, 1977.

30. McVicar JW, Richmond JY, et al. Observation of cattle, goats and pigs after administration of synthetic interferon inducers and subsequent exposure to foot and mouth disease virus. Can J Comp Med 37:362-368, 1973.

31. Georgiades J. Effect of low dose natural human interferon alpha given into the oral cavity on the recovery time and death loss in feedlot hospital pen cattle: a field study. Arch Immunol Ther Exp 41(3-4):205-207, 1993.

32. Cummins JM, Hutcheson DP. Oral therapy with human interferon alpha in calves experimentally injected with infectious bovine rhinotracheitis virus. Arch Immunol Ther Exp 41(3-4):193-197, 1993.

33. Cummins JM, Guthrie D, et al. Natural human interferon-" administered orally as a treatment of bovine respiratory disease complex. J Interferon Cytokine Res 19(8):907-910, 1999.

34. Young AS, Maritim AC, et al. Low-dose oral administration of human interferon alpha can control the development of Theileria parva infection in cattle. Parasitology 101 Pt 2:201-209, 1990.

35. Chinsangaram J, Moraes M, et al. Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease. J Virology 77(2):1621-5, 2003.
 
36. Wu Q, Mario CS, et al. Adenovirus - mediated Type 1 interferon expression delays and reduces disease signs in cattle challenged with foot-and-mouth disease virus. J Interferon Cyto Res 23:359-368, 2003.

37. Williams BR. PKR; a sentinel kinase for cellular stress. Oncogene 18(45):6112-6120, 1999.

38. Tan SL, Katze MG The emerging role of the interferon-induced PKR protein kinase as an apoptotic effector: a new face of death? J Interferon Cyto Res 19:543-554, 1999.

39. Brown CC, Piccone ME, et al. Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle. J Virol
70(8):5638-5641, 1992.



Dr. Joseph M. Cummins

Amarillo Biosciences, Inc.

4134 Business Park Drive

Amarillo, Texas 79110 USA

Tel: 806-376-1741

Fax: 806-376-9301

E-mail:  <mailto:jcummins@amarbio.com> jcummins@amarbio.com

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