: Perdue Study autoantibodies from vaccines

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Ok, here is the study.There is other provocative articles stated this that are

even much older if you are interested.Sincerely,Patricia Jordan DVM,CVA,CTCVM &

Herbology

 

 

FW: Perdue StudySat, 11 Aug 2007 16:04:22 -0400

 

 

Sincerely,

Patricia Monahan Jordan, DVM,CVA,CTCVH & Herbology

 

 

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Perdue StudySat, 10 Mar 2007 21:14:31 -0500

 

 

 

Vaccine-Induced Autoimmunity in the Dog

Advances in Veterinary Medicine Vol 41, pp 733-747

 

 

 

 

 

HARM HOGENESCH, JUAN AZCONA-OLIVERA, CATHARINE SCOTT-MONCRIEFF, PAUL W. SNYDER,

AND LARRY T. GLICKMAN

Departments of Veterinary Pathobiology and Veterinary Clinical Sciences, Purdue

University, West Lafayette, Indiana 47907

 

 

I. IntroductionII. Materials and Methods A. Animals B. Vaccination

Schedule C. Viral Serology D. Hematology E. Endocrinology F.

Immunology G. Lymphocyte Blastogenosis Assay H. Enzyme-Linked

Immunosorbent Assay (ELISA) I. Necropsy J. Statistical AnalysisIII.

Results A. Viral Serology B. Clinical Observations, Hematology, and

Endocrinology C. Immunology D. NecropsyIV. Discussion

Acknowledgements References

 

 

 

 

 

 

I. Introduction

Vaccines are widely used in human and veterinary medicine as an effective and

economic method to control viral and bacterial diseases. Although generally

considered safe, vaccination is occasionally accompanied by adverse affects.

Many adverse affects related to vaccination are acute and transient, for

example, fever, swelling at the site of the inoculation, and allergic reactions.

In contrast, reports of autoimmune disease following vaccination are relatively

rare. In most instances, it is difficult, if not impossible, to ascertain that

vaccination caused or precipitated the autoimmune disease. In a recent report,

the Advisory Committee on Immunization Practices in people concluded that there

is a causal relation between diptheria-tetanus-pertussis (DTP) and

measles-mumps-rubella (MMR) vaccination and arthritis, but no evidence of a

causal relationship between these vaccinations and other autoimmune diseases

such as autoimmune hemolytic anemia and Guillain-Barre syndrome (Centers for

Disease Control and Prevention, 1996). Cohen and Shoenfeld (1996) also stated

that the relation between vaccination and autoimmunity is obscure. They added

that there is a need for experimental studies to address this subject (Cohen and

Shoenfeld, 1996).

There has been a growing concern among dog owners and veterinarians that the

high frequency with which dogs are being vaccinated may lead to autoimmune and

other immune-mediated disorders (Dodds, 1988; Smith, 1995). The evidence for

this is largely anecdotal and based on case reports. A recent study observed a

statistically significant temporal relationship between vaccination and

subsequent development of immuno-mediated hemolytic anemia (IMHA) in dogs (Doval

and Ciger, 1996). Although this does not necessarily indicate a causal

relationship, it is the strongest evidence to date for vaccine-induced

autoimmune disease in the dog.

We are investigating the effect of vaccination on dogs in a series of

experimental studies. The goals of these experiments are (1) to determine if

vaccination of dogs affects the function of the immune system and, in

particular, if vaccination results in autoimmunity; (2) to delineate the

mechanisms by which vaccination results in autoimmunity if this occurs; and (3)

to develop alternative vaccination strategies that will not be accompanied by

adverse effects. The issue that is the focus of this and ongoing studies in our

laboratory is somewhat different from that examined by Duval and Ciger (1996).

In their study, a statistically significant temporal relationship between the

onset of IMHA and prior vaccination suggested that vaccination caused IMHA or

accelerated preexisting IMHA in adult dogs. Although not documented, it is

likely that these middle-aged dogs had received multiple vaccines prior to the

last vaccination. Why this last vaccination suddenly triggered the onset of IMHA

is unknown. In contrast, our studies examine if vaccination of dogs at a young

age causes alterations in the immune system, including the production of

autoantibodies, that could eventually lead to autoimmune disease in susceptible

individuals. In this paper, we report on the findings of the first study in

which a group of vaccinated dogs and a group of unvaccinated dogs were followed

for 14 weeks after the first vaccination.

II. Materials and Methods

A. Animals

Two pregnant Beagle dogs were purchased from a commercial breeder. The animals

whelped in the Animal Facility of the Purdue University School of Veterinary

Medicine and the pups were weaned at 6 weeks of age. Five pups were assigned to

one of two groups, a vaccinated and an unvaccinated group, based on body weight,

gender, and litter of origin. The vaccinated and unvaccinated group of dogs were

housed in separate rooms.

The dogs were examined daily. Rectal temperature and body weight were recorded

twice a week. Blood samples were collected from the jugular vein prior to each

vaccination and 2, 5, 7, and 14 days following vaccination for hematology,

endocrinology, and viral serology. Blood samples collected on days 5 and 14

following vaccination were also used for lymphocyte phenotyping and lymphocyte

proliferation assays, and blood samples collect at 7 days following vaccination

were used for the detection of autoantibodies.

B. Vaccination Schedule

The dogs in the vaccinated group were injected subcutaneously with a

commercially available multivalent vaccine, Vanguard-5 CV/L (Pfizer, Croton, CT)

at 8, 10, 12, 16, and 20 weeks of age according to the instructions of the

manufacturer. They were injected subcutaneously with an inactivated rabies

vaccine, Imrab-2 (Rhone-Mericux, GA) at 16 weeks of age. The unvaccinated group

of dogs received subcutaneous injections of sterile saline at the same time

points.

Both groups of dogs were injected subcutaneously with 1 mg of keyhole limpet

hemocyanin (KLH, Calbiochem) in RIBI-adjuvant at week 20.

C. Viral Serology

Serum samples collected at 6 weeks of age and 0, 2, 5, 7, and 14 days after each

vaccination were assayed for the presence of antibodies to canine distemper

virus by serum neutralization test, and for antibodies against canine parvovirus

by hemagglutination inhibition test. Serum samples were analyzed for antibodies

against rabies virus at 16 and 20 weeks of age by a rapid fluorescent focus

inhibition test.

D. Hematology

Blood samples were collected at 0, 2, 5, 7, and 14 days after each vaccination

for hematocrit, corrected white blood cell count and differential, and platelet

counts.

E. Endocrinology

Plasma and serum samples collected at 0, 2, 5, 7, and 14 days after each

vaccination were assayed for curtisol, triiodothymonine (T3), and thyroxine (T4)

by radioimmunoassay.

F. Immunology

Lymphocyte phenotyping was used. Whole blood was stained with a panel of mouse

monoclonal antibodies, followed by F(ab')2 goat anti-mouse IgG (Jackson Research

Laboratories). The monoclonal antibodies used were CA2.1D6 (anti-CD21), CA15.8G7

(anti-TCRoB), CA20.8H1 (anti-TCRv81, 12.125 (anti-CD4), and 1.140 (anti-CD8).

The characteristics of these monoclonal antibodies have been described (Gebhard

and Carter, 1992; Moore et al., 1995). Following red blood cell lysis and

fixation in 2% paraformaldehyde, the cells were analyzed by flow cytometry.

G. Lymphocyte Blastogenosis Assay

Heparinized blood samples were diluted 1:10 in RPM1-1640 and distributed in the

wells of a 96-well plate. Triplicate samples were incubated for 96 hours in the

presence of medium only, 2.5 and 5 pg/ml PHA, 5 and 10 pg/ml Concenavalin A (Con

A) and 1 and 10 pg/ml PWM. During the last 24 hours of incubation the wells were

pubed with 0.5 uCi of H-thymidine. The cells were harvested with a 96-well cell

harvester, and the incorporation of radioactivity was measured in a TopCount

scintillation counter (Packard Instrument Co., Meriden, CT).

H. Enzyme-Linked Immunosorbent Assay (ELISA)

The presence of antibodies reactive with homologous and heterologous antigens in

serum samples collected at 22 weeks of age was analyzed by an indirect ELISA.

High-binding ELISA plates (Costar, Cambridge, MA) were coated with 10 pg/ml of

antigen in 0.1 M bicarbonate buffer. The wells were rinsed and incubated for 1

hour with phosphate-buffered saline (PBS)/0.1% Tween. Serum samples were diluted

1:10 in PBS and added to the wells in triplicate. Following incubation, the

wells were rinsed and incubated with alkaline phosphatase labeled goat anti-dog

IgG (Kirkegnard and Perry, Gaithersburg, MD). Alkaline phosphatase activity was

measured after addition of p-NPP substrate at 405 nm in a microplate reader

(Molecular Devices, Menlo Park, CA).

Essentially the same procedure was used to measure the presence of antibodies

against KLH. Alkaline phosphatase labeled anti-dog IgM and IgG were used as

secondary reagents.

I. Necropsy

At 22 weeks of age, the dogs were killed by intravenous injection of

barbiturates, and a complete necropsy performed. Tissue samples were collected

in 10% buffered formalin and processed for light microscopic examination. The

tissues that were examined included the spleen, lymph nodes, tonsils, thymus,

Psyer's patches, adrenal glands, thyroid glands, pituitary gland, pancreas,

heart, lung, kidney, liver, and brain.

J. Statistical Analysis

Data were analyzed for significant differences between groups by Student's t

test or repeated measures ANOVA and a significant change over time using a

repeated measures ANOVA.

III. Results

A. Viral Serology

None of the pups had detectable antibodies against canine distemper virus and

canine parvovirus at 6 weeks of age and against rabies virus at 16 weeks of age.

The unvaccinated dogs remained seronegative for these three viruses during the

course of the study. The dogs that were immunized developed titers against CDV

(maximum titers ranged from 1:48 to 1:1024), CPV-2 (1:320 to 1:1280), and rabies

(1:25 to 1:1000).

B. Clinical Observations, Hematology, and Endocrinology

No differences between the unvaccinated and vaccinated groups were found for

rectal temperature, body weight, and hematologic values.

There were no significant differences between unvaccinated and vaccinated dogs

for concentrations of cortisol, T3, and T4. However, a significant (p<0.02)

change was observed over time for each of these three hormones. The plasma

concentration of cortisol decreased from a mean of 41.1 ng/ml at 8 weeks of age

to 17.6 ng/ml at 22 weeks of age. The concentration of T4 also decreased, from

31.1 ng/ml at 8 weeks of age to 22.8 ng/ml at 22 weeks of age. The concentration

of T3 increased from 0.63 ng/ml at 8 weeks of age to 1.1 ng/ml at 22 weeks of

age.

C. Immunology

No differences were observed unvaccinated and vaccinated dogs for lymphocyte

subpopulations or for the proliferative response to any of the mitogens tested.

The response of both groups of dogs to KLH was similar. There was no

statistically significant difference in the KLH-specific IgM and IgG

concentrations in the serum (not shown).

At 8 weeks of age, antibodies against homologous and conserved heterologous

antigens were negligible in the serum of the dogs. At 22 weeks of age there was

a significant increase of IgG antibodies reactive with 10 of 17 antigens in the

vaccinated dogs versus no increase in the unvaccinated dogs (Table I). The

increase of optical density was modest for 8 of these 10 antigens, but a large

increase was observed for fibronectin and laminin. All vaccinated dogs developed

high levels of fibronectin-specific IgG antibodies. Similar levels of IgG

anti-fibronectin antibodies were observed when bovine fibronectin was

substituted by human or mouse fibronectin (not shown). The concentration of

anti-fibronectin antibodies began to increase after the second vaccination in

three dogs and after the third vaccination in the other two vaccinated dogs, and

reached a maximum level after the fourth vaccination (Fig. 1). To determine if

the antibodies had a preferential reactivity with a particular part of the

bironectin molecule, we tested the reactivity of serum samples with two

fragments of the fibronectin. The 30-kDa fragments contains the heparin-binding

domain of fibronectin, whereas the 45-kDa fragment contains the collagen-binding

domain. As shown in Fig. 2, little reactivity was observed with the 45-kDa

fragment, but significant reactivity was observed with the 30-kDa fragment.

High levels of anti-laminin antibodies were observed in the serum of three of

the five vaccinated dogs at 22 weeks of age. One dog had high levels at 17 weeks

of age, whereas the other two dogs did not devlop high levels until the end of

the study.

High levels of antibodies reactive with skeletal muscle myosin and myoglobin

were observed in both groups of dogs at 22 weeks of age. The antibody levels

increased at 11 weeks of age in three dogs, at 13 weeks of age in another three

dogs, and at 17 weeks of age in the remaining four dogs.

D. Necropsy

Gross and light microscopic examination of the tissues of the dogs revealed no

significant lesions. The thyroid gland of one of the vaccinated dogs had a small

lymphoid nodule with obliteration of adjacent thyroid follicles.

IV. Discussion

In this study, we exhaustively evaluated the effects of vaccination with a

multivalent vaccine and a rabies vaccine on the immune system of young dogs.

Vaccination did not cause immunosuppression or alter the response to an

unrelated antigen (KLH). In contrast to an earlier study (Mastro et al., 1986),

but in agreement with other work (Phillips and Schultz, 1987), we did not

observe a transient lymphopenia in the dogs at any time. However, vaccination

did induce autoantibodies and antibodies to conserved heterologous antigens. The

pathogenic significance of these autoantibodies is presently uncertain. We did

not find any evidence of autoimmune disease in the vaccinated dogs, but the

study was terminated when the dogs were 22 weeks of age, well before autoimmune

diseases usually become clinically apparent. It is likely that genetic and

environmental factors will trigger the onset of clinical autoimmune disease in a

small percentage of the animals that develop autoantibodies. For practical and

economic reasons, only a small number of dogs can be followed in an experimental

study, and clinical autoimmune disease may, therefore, never be observed. The

principal value of an experimental study is that it enables us to determine the

frequency of autoantibody responses and the mechanism(s) that cause vaccines to

induce autoantibodies.

We used two vaccines, a multivalent vaccine and an inactivated rabies vaccine of

a particular commonly used brand. We consider it unlikely that the observed

autoantibodies were specifically induced in response to those brands of vaccine

and this phenomenon will likely occur with other commercial vaccines. In a

follow-up study, we have observed similar autoimmune phenomena in dogs immunized

with the multivalent vaccine only and in dogs immunized with the rabies vaccine

only (unpublished observations).

There was a marked increase of autoantibodies to the skeletal muscle proteins,

myoglobin and myosin, in both groups of dogs. The reason for the appearance of

these antibodies is uncertain, but it may be the result of the frequent blood

sampling of the dogs. The dogs were bled five times following each vaccination,

and some tissue trauma was unavoidable.

We examined the thyroid and adrenal cortical function in the dogs, and did not

find evidence of any abnormality. Autoimmune thyroiditis is one of the most

common autoimmune diseases of dogs, and it has been suggested that the apparent

increase of this condition in dogs is related to the increased frequency of

vaccination with modified live vaccines. There was no increase of

anti-thyroglobulin antibodies in the vaccinated animals, or other evidence of

thyroid dysfunction. However, the lymphoid nodule found in the thyroid gland of

one of the vaccinated dogs may be an early manifestation of thyroiditis, a

common lesion in purpose bred Beagles (Fritz et al., 1970).

The most strikingly increased concentrations of autoantibodies were directed

against fibronectin and laminin. Fibronectin is widely distributed in the body

as a component of the extracellular matrix and plasma. The anti-fibronectin

antibodies were reactive with fibronectin of bovine, murine, and human origin.

Although we have not yet demonstrated that they also react with canine

fibronectin, this is very likely, since fibronectin is highly conserved between

species. Anti-fibronectin antibodies have been found in human patients with

systemic lupus erythematosus (SLE) and rheumatoid arthritis, and a patient with

a poorly defined connective tissue disease (Henane et al., 1986; Atta et al.,

1994, 1995; Girard et al., 1995). The anti-fibronectin antibodies in four human

SLE patients were directed against the collagen-binding domain (Atta et al.,

1994), in contrast to the anti-fibronectin antibodies in the vaccinated dogs,

which showed no affinity for this domain. The anti-fibronectin antibodies in the

human patient with connective tissue disease showed reactivity with the

cell-binding domain of fibronectin (Girard et al., 1995).

Anti-fibronectin antibodies have been experimentally induced in rabbits by

immunization with human fibronectin in complete Freund's adjuvant

(Murphy-Ullrich et al., 1984). The antibodies were reactive with both human and

rabbit fibronectin. The rabbits subsequently developed a glomernlopathy with

granular deposits suggestive of immune complexes in the glomcrular basement

membrane. Anti-fibronectin antibodies have been induced in mice by multiple

injections of homologous fibronectin without adjuvant (Murphy-Ulrich et al.,

1986). The titer of anti-fibronectin antibodies was much lower in mice immunized

with native fibronectin than in mice immunized with de-natured fibronectin.

However, in both groups, immune complexes were present in the serum and in the

glomerali (Murphy-Ullrich et al., 1986). Light microscopic examination of the

glomerali of the kidneys of vaccinated dogs did not reveal evidence of

glomerunfpathy, but we cannot exclude the possibility of sub-light microscopic

lesions.

Anti-laminin antibodies were prevalent in the serum of three of the five

vaccinated dogs. Anti-laminin antibodies are increased in human patients with

SLE, rheumatoid arthritis, and vasculitis. Injection of polyclonal anti-laminin

antibodies into rats resulted in glocnerulopathay and proteinuria (Abrahamson

and Caulfield, 1982)> Anti-laminin antibodies have also been implicated in

glomaerular disease in rats induced by mercuric chloride (Aten et al., 1995).

The mechanisms that may underlie the production of autoantibodies following

vaccination are unknown, but at least four mechanisms can be proposed:

cross-reactivity with vaccine-components, somatic mutation of immunoglobulin

variable genes, " bystander activation " of self-reactive lymphocytes, and

polyclonal activation of lymphocytes. Perhaps the simplest and most likely

mechanism is that of cross-reactivity of vaccine and self-antigens. (Schattner

and Rager-Ziaman, 1990), the most likely sources of cross-reactive epitopes are

bovine serum and cell culture components. These are present in almost all

vaccines as residual components of the cell culture necessary to generate

vaccine viruses and may purposely be added to the vaccine as a stabilizer. In

the presence of an adjuvant, these bovine products stimulate a strong immune

response and induce antibodies that cross-react with conserved canine antigens.

Thus, the strong response to fibronectin in the vaccinated dogs is most likely

the result of the injection of bovine fibronectin contaminants in the vaccine.

Indeed, this is essentially identical to the protocol used to produce

anti-fibronectin antibodies in rabbits with human fibronectin in complete

Freund's adjuvant (Murphy-Ullrich et al., 1984), as mentioned above. The lower

response to other antigens (e.g., cardiolipin and laminin) may be due to a lower

concentration of these antigens in the vaccine or lower immunogenicity.

During every immune response, self-reactive B and T lymphocytes are generated

and activated. This is the result of somatic mutation and bystander activation.

Under normal conditions, this will not lead to significant production of

autoantibodies, because of the selection process in the germinal centers of

lymph nodes. In the germinal centers only B cells that successfully compete for

interaction with antigen presented on the surface of follicular dendritic celia

will be allowed to survive (MacLennan, 1994). These B cells generally have

high-affinity receptors for the antigen to which the immune response was

induced. B cells with low affinity for the antigen or affinity for other

antigens, including self-antigens, will undergo programmed cell death. The B

cells with high-affinity receptors express bc1-2, which may rescue them from

programmed cell death (MacLennan, 1994). This mechanism was elegantly

demonstrated in mice immunized with a nominal antigen phosphorylcholine (Ray et

al., 1996). A single point mutation in the hypervariable region of the expressed

immunoglobulin genes was sufficient for the phosphorylcholine-specific B cells

to acquire specificity for DNA. However, it was only possible to demonstrate

DNA-specific B cells by fusing germinal center B cells with celia that expressed

high levels of bc1-2, thereby rescuing them from programmed cell death (Ray et

al., 1996). An increased expression of bc1-2 was observed in thymic lymphoid

follicles of patients with myasthenia gravis, suggesting that failure to delete

self-reactive B cells in these patients may lead to autoimmune disease (Shiono

et al., 1997). While this may seem an attractive hypothesis to explain

autoimmune phenomena in human beings and dogs, there is currently no evidence

that this is a common mechanism.

Finally, polyclonal activation of lymphocytes, including activation of

self-reactive lymphocytes, is a possible mechanism of vaccine-induced

autoimmunity. Certain viruses and bacteria have superantigen or mitegen activity

(Schwarts, 1993). This could also be the case for the microbial products

included in the vaccines. The present study does not support this mechanism.

Firstly, antibodies were observed against 10 of 17 antigens tested. Secondly,

the anti-fibronectin antibodies did not react with any portion of the

fibronectin molecule, but instead, reacted most strongly with the heparin

binding domain. These observations indicate that the appearance of

autoantibodies in the serum of vaccinated dogs is an antigen-driven process and

not caused by polyclonal activation. As argued earlier, the main antigens

implicated are cell culture contaminants and bovine serum components.

In the dog, certain autoimmune diseases occur more frequently in particular

breeds of dogs, indicating genetically determined susceptibility (Dodds, 1983;

Happ, 1995). There is abundant evidence from studies in rodents and human beings

that the magnitude of the antibody response and the susceptibility to autoimmune

disease are in part genetically determined (Schwartz, 1993). It is likely that

genetic factors also determine the susceptibility to vaccine-induced

autoimmunity. That this is indeed the case is suggested by the finding that only

three of the five vaccinated dogs developed a strong anti-laminin antibody

response and that the kinetics of the anti-fibronectin response differed between

individual animals. Identification of susceptibility genes will be important,

because it may shed light on the pathogenesis of the autoimmunity. In addition,

it will provide genetic tests that will enable dog breeders to monitor the

susceptibility of their breeding stock to vaccine-induced autoimmunity.

Although the pathogenic significance of the vaccine-induced autoantibodies is

still unclear, there are a number of ways to prevent their induction. Not

vaccinating dogs is not a viable option, because the benefits of vaccination

clearly outweigh the still uncertain risks of immune-mediated disease. However,

since bovine serum components in the vaccine may be responsible for the majority

of autoantibodies, elimination of these bovine components may avoid this

problem. This could be accomplished by substituting homologous serum for bovine

serum. However, as mentioned earlier, anti-fibronectin antibodies may still be

induced by immunization with homologous fibronectin. New generations of

vaccines, especially naked DNA vaccines, are free of serum components, and these

should not induce autoantibodies. A recent study in mice indicates that DNA

vaccination does not induce or accelerate autoimmune disease (Mer et al., 1997).

Finally, mucosal vaccines are less likely to induce autoantibodies than

parenterally administered vaccines. Depending on the formulation of the vaccine,

soluble serum components are less likely to be absorbed via the mocosal surface,

and, in fact, may induce tolerance instead of autoantibodies (Weiner et al.,

1994).

In conclusion, we have demonstrated that vaccination of dogs using a routine

protocol and commonly used vaccines, induces autoantibodies. The autoantibody

response appears to be antigen driven, probably directed against bovine antigens

that contaminate vaccines as a result of the cell culture process and/or as

stabilizers. The pathogenic significance of these autoantibodies has not yet

been determined.

Acknowledgments

The authors thank Cheryl Anderson and Julie Tobelski-Crippen for animal care and

technical support, and Nita Glickman for data management. This work is supported

by the John and Winifred Hayward Foundation.

 

 

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