Compounds for the prevention and treatment of helmith infections

A compound for protecting a vertebrate against infection by helminths comprising hyaluronidase covalently coupled to an immunostimulating carrier, in preferred form, the immunostimulating carrier to which the hyaluronidase is covalently coupled comprising a copolymer of ethylenepiperazine N-oxide and N-ethylacetylethylenepiperazinium bromide (hereafter referred to as "SYNPOL"), and a process for protecting a vertebrate against infection by helminths comprising administration to the vertebrate of a therapeutically effective amount of a compound comprising hyaluronidase covalently coupled to an immunostimulating carrier, preferably SYNPOL, in preferred form, the compound being administered to the vertebrate in the form of a vaccine.

FIELD OF THE INVENTION 
The present invention relates to compositions capable of eliciting an 
immune response in vertebrates against helminth infections. In particular, 
the present invention is directed to vaccines which may be used to protect 
a vertebrate against infection from parasitic helminths. 
Helminthic infections are a major cause of morbidity and mortality in both 
domesticated animals and human populations. Speaking generally, helminths 
refer to parasitic and non-parasitic species belonging to the phyla 
platyhelminthes (for example, flukes, tapeworms, and other flatworms) and 
nematahelminthes (for example, roundworms and their relatives). The 
following is illustrative of how helminthic infections occur. A helminth 
species enters the body of a host in the form of eggs or invasive larvae, 
for example, as a result of the ingestion by the host organism of food 
containing the eggs or larvae. The helminths then develop, moving slowly 
through different tissues, blood and/or lymph. Finally, they reach their 
"preferred" organ, and grow and mature. Eventually, the organ in which 
they reside is affected adversely. 
Control measures rely to a large extent on improvements in hygiene, 
reduction in vector populations, and chemotherapy. Control measures which 
focus on hygiene and reduction of vector population have proved to be 
problematic especially in developing countries where such infections are 
most prevalent and such control measures are most difficult to implement. 
Current chemotherapeutic medications all have drawbacks such as high 
toxicity, the requirement that treatments be repeated and 
immunosuppressive side-effects. Furthermore, the use of these preparations 
often results in the parasites' developing resistance to the 
chemotherapeutic medication. Indeed, nearly every country has documented 
cases of antihelminthic resistance. For these reasons, as well as the 
expense of repeated administration of chemotherapeutic compounds, a 
compound which would not have these drawbacks has been highly sought 
after. 
Since a large variety of helminth species may be found in infected 
populations, it would desirable to produce a vaccine having a broad 
spectrum of prophylactic activity. To date, the induction of strong 
protective host immunity following infection by helminths has been 
uncommon. The long co-evolutionary experience these parasites have had 
with their hosts has driven the host-parasite relationship to a level of 
accommodation that results in chronic or persistent conditions rather than 
acute infections that typically yield strong specific immunity. For this 
reason, unlike the situation with most microbial diseases of animals, few 
vaccines have been available for helminth control, and those which do 
exist have significant drawbacks. 
Of the known methods of producing helminth vaccines, inactivated and living 
vaccines have the drawback that they are labor-intensive and only weakly 
immunogenic and they induce side-effects such as a localized inflammation, 
allergic response, and fever. Furthermore, often the attenuated form used 
in the vaccine causes a disease similar to that induced by the virulent 
(wild) forms of the parasite. In addition, the macromolecular carrier 
proteins used in these vaccine preparations cause a number of 
immunopathologic side-effects in the vaccinated organism. 
The other available class of vaccines comprises genetically engineered 
antigens. Yet these too are only weakly immunogenic. 
In summary, the available types of vaccines are generally ineffective and 
possess a narrow specificity, being directed against a single parasite. 
REPORTED DEVELOPMENTS 
A detailed description of helminths in whose life-cycles tissue-migration 
plays an important part, as well as a discussion of the pathological 
conditions they cause can be found for example in E. J. Soulsby, Helminth, 
Arthropods and Protozoa of Domesticated Animals, 7th Edition, Lea and 
Febiger, Philadelphia (1982) and in G. M. Urquhart, "Veterinary 
Parasitology", Longman Scientific and Technical, United Kingdom (1987). 
All parasites elicit immune responses, but for many reasons are able to 
present a moving and sometimes invisible target to the host's immune 
response, to such an extent that the normal control mechanisms fail and 
immunological damage instead of immunity often occurs. This in turn 
frequently leads the host to switch off its ineffective and often 
counterproductive immune response, thereby resulting in gross pathological 
changes and immunosuppression. 
The structural and antigenic diversity of the parasitic helminths is 
reflected in the heterogeneity of the specific immune responses they 
elicit. Parasitic helminths often evade the immune system by masking and 
shedding their surface antigens and by varying their antigens during their 
residence in vertebrate hosts. This ability to mask, shed and vary surface 
antigens is a primary cause of the difficulty experienced heretofore in 
producing efficacious vaccines against helminths infection. 
A review of modern vaccines used in the treatment of parasitic diseases is 
provided in, J. H. L. Playfair. et al., The Lancet 335 (1990): 1263-1266, 
while a more general discussion of the nature of the immunological 
response of hosts to parasitic helminths can be found in the article by S. 
Lloyd and E. J. L. Soulsby in "Parasitology in Focus: Facts and Trends", 
Ed. H. Mehlhorn, Springer-Verlag (1988) pp. 619-650. As indicated in the 
Playfair et al. review article and as mentioned hereinabove, since 
existing helminth control measures are expensive and difficult to 
implement on a wide scale, there is a strong need for vaccines capable of 
reducing the intensity and prevalence of helminth infection in host 
populations. 
Prior to the present invention, it was thought unlikely that one antigen 
alone could confer adequate protection against a wide range of helminth 
infections based on the difficulties referred to above encountered in 
producing effective anti-helminth vaccines against even specific species. 
For an overall review of medical and scientific challenges provided by 
helminths, see A. A. F. Mahmoud, Science 246 (1989): 1015-1021. (In 
addition see also the entries "Parasites, Escape from Immunity", by D. J. 
Mclaren, and "Parasites, Immunity to" by F. E. G. Cox, in the Encyclopedia 
of Immunology, eds. I. M. Roitt and P. J. Delves, Academic Press, 1992.) 
SUMMARY OF THE INVENTION 
In accordance with the present invention, there is provided a compound for 
protecting a vertebrate against infection by helminths comprising 
hyaluronidase covalently coupled to an immunostimulating carrier. In 
preferred form, the compound of the present invention is administered to 
the vertebrate in the form of a vaccine and the immunostimulating carrier 
to which the hyaluronidase is covalently coupled comprises copolymers of 
ethylenepiperazine N-oxide and N-ethylacetylethylenepiperazinium bromide 
(hereafter referred to as "SYNPOL"). 
The present invention is based in part on the discovery that the enzyme 
hyaluronidase is produced by larval helminth species in order to penetrate 
the tissue barriers in the body of a host organism they have infected. In 
practice, the present invention is capable of eliciting an immune response 
against hyaluronidase, as produced by the helminths, thereby drastically 
reducing, if not eliminating, the ability of the helminths to penetrate 
tissue barriers and thereby infect an animal. 
Another aspect of the present invention is the provision of a process for 
protecting a vertebrate against infection by helminths comprising 
administration to the vertebrate of a therapeutically effective amount of 
a compound comprising hyaluronidase covalently coupled to an 
immunostimulating carrier, preferably synpol. In preferred form, the 
compound is administered to the vertebrate in the form of a vaccine which 
includes, in admixture with the compound, synpol. Examples of vertebrates 
that can be treated in accordance with the present invention include man, 
cattle, sheep, swine, dogs, horses, cats, goats, buffaloes, camelidae and 
poultry. 
An additional aspect of the present invention is the provision of a process 
comprising reacting hyaluronidase and an immunostimulating carrier under 
conditions of time and temperature to covalently couple hyaluronidase and 
said immunogenic carrier. 
The present invention is believed to offer a variety of advantages over 
prior art techniques for protecting vertebrates from helminthic 
infections. The compound of the present invention provides protection 
against infection by those helminth species which utilize hyaluronidase to 
facilitate their migration within the body of a host organism. 
Accordingly, the compound of the present invention is efficient against a 
broad spectrum of helminth species. This is a significant advantage over 
prior art vaccines which are often limited to protecting a host from only 
one specific helminth species. 
An additional advantage offered by the vaccines of the present invention is 
that they are strongly immunogenic. Prior art anti-helminth vaccines are 
often only weakly immunogenic and induce undesirable side effects such as 
localized inflammation, allergic response, and fever. In addition, prior 
art vaccines often utilize macromolecular carrier proteins that cause a 
number of immunopathologic side effects in the vaccinated organism. 
These and the various other disadvantages of the prior art anti-helminth 
vaccines have been overcome by the provision of the compound of the 
present invention inasmuch as it is able to elicit a strong immunogenic 
response without the side effects often seen in prior art anti-helminthic 
vaccine preparations. Furthermore, the compounds of the present invention 
do not appear to effect development or fertility. 
The ability of the vaccine of the present invention to elicit a strong 
immunogenic response and to provide a broad spectrum of protection against 
a variety of helminth species offer advantages heretofore unseen in the 
art of anti-helminthic vaccines.

DETAILED DESCRIPTION OF THE INVENTION 
One aspect of the present invention is the provision of a material which 
comprises the reaction product of hyaluronidase and an immunostimulating 
carrier. 
Hyaluronidase, as the term is used herein, refers to a family of enzymes 
which hydrolyze naturally occurring polysaccharides, in particular, 
hyaluronic acid and glycosaminoglycans such as chondroitin sulfates (4-, 
6-, D and E). These polymeric substances are essential components of the 
semisolid gel-like structure of the extracellular matrix. Hyaluronidase 
cleaves these polymeric substances and therefore is capable of destroying 
extracellular matrices. A large variety of helminth species produce and 
use hyaluronidase to hydrolyze the aforementioned components of the host 
organism's extracellular matrices thereby permitting the helminths to 
penetrate tissue barriers and migrate within the body of a host organism 
until they reach a preferred organ where they grow and mature. 
The hyaluronidase family includes related enzymes which hydrolyze the 
aforementioned type substrates. These can be grouped according to their 
specificity for different linkages within the structure of hyaluronic acid 
polymer molecules. In particular, the hyaluronidases may be grouped into 
three primary groups: hyaluronoglucosaminidases, hyaluronoglucuronidases, 
and glucoronate lyases (B. Fiszer-Szafarz, Analytical Biochemistry, vol. 
143, p. 76 (1984)). 
Native hyaluronidase alone demonstrates extremely weak immunogenicity and 
does not induce visible anti-hyaluronidase antibody production, even after 
repeated injections. 
Hyaluronidase has been isolated from a variety of sources, including snake 
and bee venoms, leech saliva, the acrosomal granula of spermatozoa, the 
lysosomal granula of various cells and from bacterial toxins. Exemplary 
sources of hyaluronidase that can be used in the practice of the present 
invention include cattle and sheep testes, helminths, leeches, bee and 
snake venoms and bacteria. 
Bacterial sources of hyaluronidase include, but are not limited to, the 
following: Streptococcus millery (P. F. Unsworth, J. Clin. Pathology, 
(London) 1989, 42(5), 506-510); Streptococcus pyogenes (W. L. Hynes and J. 
J. Ferretti, Infection and Immunity, 1989, 57(2), 533-539); Streptococcus 
equisimilis (R. Sting et al., Med. Sci. Res., 1989, vol. 17, No. 17, pp. 
723-725); Clostridium difficicle (S. V. Seddon et al., J. Med. Microbiol., 
1990, v. 31, no. 3, pp. 169-174); Streptococcus uberis (P. Schaufuss et 
al., Zentralbl. Bakteriol. FRG, 1989, v. 271, no. 1, pp. 46-53); and 
Streptococcus dysgalactiae (A. Hamai et al., Agric. Biol. Chem., 1989, v. 
53, No. 8, pp. 2163-2168). In addition, yeast of the genus candida have 
also been found to contain hyaluronidase. M. T. Shimizu, Rev. Microbiol., 
1988, v. 19, No. 4, pp. 442-445). 
In a given species, hyaluronidase generally can be found in monomeric as 
well as oligomeric forms, with, for example, dimers and tetramers of the 
same subunit often being present. The amino acid sequence for 
hyaluronidase produced by streptococcus pyogenes bacteriophage has been 
determined (W. L. Hynes et al., Infection and Immunity 57 (1989): 533-539) 
and bee venom hyaluronidase has recently been sequenced (M. Gmachl et al., 
Proc. Natl. Acad. Sci. USA, 90, 3569-3573 (1993)). It is anticipated that 
the sequencing and cloning of the genes encoding hyaluronidase will be the 
basis for recombinant DNA based production of hyaluronidase for use in the 
practice of the present invention. 
A variety of commercially-available preparations of hyaluronidase may be 
used to prepare the compound of the present invention, including, for 
example, a bovine preparation of hyaluronidase sold by REANAL CO. (Catalog 
No. 0705). Polyacrylamide gel electrophoresis (PAGE) of the hyaluronidase 
obtained from this source indicates the presence of a major protein band 
having an approximate molecular weight of 63 kilodaltons (kDa). There can 
be used also a material obtained from sheep testes sold by Sigma Chemical 
Co. (catalog no. H2126). Polyacrylamide gel electrophoresis of the 
hyaluronidase obtained from this source reveals a major protein band 
having an approximate molecular weight of approximately 39 kDa. 
Hyaluronidase may also be obtained from Serva (Catalog No. 25119 and 
Catalog No. 25121). 
While the hyaluronidase preparations obtained from various commercial 
sources differ with regards to the predominate protein species present as 
evidenced by PAGE, it has been found that the various commercial 
preparations of hyaluronidase are all enzymatically active and are also 
immunologically cross-reactive with each other. In addition, practically 
all preparations investigated contain at least traces of a protein species 
having a molecular weight of approximately 60-69 kDa and one cannot 
exclude the possibility that the shorter polypeptide chains present in the 
reducing conditions used in the PAGE process are assembled under 
physiological conditions into oligomers of about 60-90 kDa. 
It is anticipated that compounds of the present invention utilizing 
hyaluronidase isolated from both ram and bull testes may offer a 
combination of particularly desirable immunogenicity, cost effectiveness, 
and convenience. Purification methods for such hyaluronidases are rather 
well developed and include the commonly used steps of extraction, 
precipitation, centrifugation, ultrafiltration, ion exchange, and gel 
chromatography. The compounds of the present invention utilizing 
hyaluronidase isolated from sheep or bovine testes provoke an immune 
response to helminth hyaluronidase. Utilization of hyaluronidase isolated 
from sheep or bovine testes is also significantly more cost-effective than 
isolating hyaluronidase from helminth larvae. Hyaluronidase of testicular 
origin has been found to cleave hyaluronic acid and is also able to 
recognize chondroitin sulfates. (Bartolucci et al., Int. J. Tissue React., 
13(6) (1991), p. 311). Accordingly, particularly preferred embodiments of 
the compounds of the present invention are made utilizing hyaluronidase 
obtained from the testicles of rams or bulls. In this regard, it has been 
found that a hyaluronidase of sheep or bovine origin sold by Sigma 
Chemical Company is suitable in the practice of the present invention. 
It has been observed that the compounds of the resent invention which 
utilize hyaluronidase obtained from a source other than the animal 
receiving the treatment of the present invention is often more immunogenic 
than compounds utilizing hyaluronidase isolated from the species being 
treated. For example, hyaluronidase isolated from sheep tends to induce a 
stronger immune response in cattle than hyaluronidase isolated from 
cattle. For cost effectiveness and convenience in the treatment of sheep 
and cattle, consideration should be given to use of a compound that is 
prepared from a mixture of both ram and bull hyaluronidases. 
Compounds within the scope of the present invention comprise the reaction 
product of hyaluronidase, as described above, and an immunostimulating 
carrier. As the term is used herein, "immunostimulating carrier" refers to 
a compound which, when combined with a given antigen, provides for a 
highly immunogenic complex (antigen-immunostimulant) which may effectively 
immunize even low responding individuals to a given antigen. Examples of 
immunostimulating carriers which can be used in the practice of the 
present invention are described in the following publication which 
includes a discussion of the utilization of synthetic polyions as 
immunostimulators: Khaitov, R., Annals New York Academy of Sciences, 685, 
788-802, Jun. 23, 1993. Reference is made in this article to polyoxidonium 
which is equivalent to Synpol as used in the present invention. 
In preferred embodiments, the immunostimulating carrier that is reacted 
with hyaluronidase is SYNPOL. As used herein, the term "SYNPOL " refers to 
copolymers of ethylenepiperazine N-oxide and 
N-ethylacetylethylenepiperazinium bromide, corresponding to the formulae 
shown below in FIG. 1, where n=200-2000; q=(0.2-0.35)n; z=(0.4-0.65)n; 
m=(0-0.4)n. 
SYNPOL, unlike most other carriers and adjuvants, is non-immunogenic. It is 
thought that SYNPOL has no recognizable antigenic determinants and, 
accordingly, does not provoke an immune response thereby avoiding 
undesirable side effects observed with most other adjuvants and carriers 
used in vaccine preparations. 
In order to identify in a convenient way the various species of SYNPOL one 
from another, the term "synpol" is used in combination and sequentially 
with values for each of the aforementioned letters "n", "q", "z", and "m". 
For example, ethylenepiperazine N-oxide and N-acetylethylenepiperazinium 
bromide with n=1000, q=0.35, z=0.60, m=0.05 is referred to as "SYNPOL 
1000-35/60". 
An example of a specific SYNPOL species copolymer used successfully as an 
immunostimulating carrier in the vaccine embodiments of the present 
invention will be referred to herein as "SYNPOL 1000-20/50". Synpol having 
a molecular weight of at least about 15 kDa or greater is preferred in the 
practice of the present invention with SYNPOL having a molecular weight 
greater than at least about 30 kDa being especially preferred. 
The compound of the present invention can be made by any suitable method 
which effects the chemical linking of hyaluronidase to the 
immunostimulating carrier, for example, by covalently coupling 
hyaluronidase to the immunostimulating carrier. Such covalent bonds can be 
formed directly between reactive groups on the hyaluronidase and on the 
immunostimulating carrier or they can be formed through one or more 
linking groups. As will be seen in examples set forth hereinbelow, a 
preferred method for preparing the reaction product of hyaluronidase and 
the preferred immunostimulating carrier of the present invention, that is, 
SYNPOL, involves use of the azide method. This method involves converting 
the acid or ester form of SYNPOL to the hydrazide by use of hydrazine and 
thereafter combining it with hyaluronidase under conditions which produce 
a reaction product in which hyaluronidase is covalently coupled to SYNPOL. 
Alternatively, and as also illustrated in the following examples, another 
preferred method for preparing the reaction product of hyaluronidase and 
SYNPOL involves the formation of a succinimide ether of synpol. The 
succinimide ether is then combined with hyaluronidase under conditions to 
produce a compound for use in the practice of the present invention. 
It is believed that the compound of the present invention will be used most 
widely to protect vertebrates from infection by helminths. For this 
purpose, it is preferred that the product of the reaction of a 
hyaluronidase and an immunostimulating carrier be used in the form of a 
vaccine. As the term is used herein, "vaccine" refers to a composition 
which contains the compound of the present invention and which is in a 
form that is capable of being administered to a vertebrate. Typically, the 
vaccine comprises a conventional saline or buffered aqueous solution 
medium in which the compound of the present invention is suspended or 
dissolved. In this form, the compound of the present invention can be used 
conveniently to prevent, ameliorate, or otherwise treat a helminth 
infection. 
In preferred form, the vaccine of the present invention additionally 
includes an adjuvant which can be present in either a minor or major 
proportion relative to the compound of the present invention. The term 
"adjuvant" as used herein refers to non-specific stimulators of the immune 
response which when combined with the vaccine of the present invention, 
provide for an enhanced immune response. A variety of adjuvants can be 
used. Examples include complete and incomplete Freund's adjuvant, aluminum 
hydroxide, and modified muramyldipeptide. In preferred embodiments of the 
present invention, SYNPOL is used as an adjuvant in admixture with the 
compound of the present invention. 
As mentioned herein above, the compounds of the present invention are 
intended to be used to protect vertebrates species from helminthic 
infections. Examples of vertebrates that can be treated in accordance with 
the present invention include man and various domesticated animals, 
including, for example, cattle, sheep, swine, dogs, horses, cats, and 
goats, as well as other equidae, buffaloes, camelidae, and poultry. In 
particular, it is expected that the compounds of the present invention 
will be efficacious in the prevention and treatment of parasitic helminth 
infections in animals which are exposed to helminth species which utilize 
hyaluronidase. 
The compound of the present invention may be administered parenterally by 
intramuscular, subcutaneous, or intradermal administration. The preferred 
route of administration for a given organism may be found by reference to 
the Examples section of the application. Preferred dose ranges may vary 
given the animal being treated and the most prevalent helminth species in 
a given environment, but in general, a vaccine dose of about 0.05 mg of 
protein/kg of animal weight has been found to be effective. Further 
guidance regarding effective does ranges may be found by referring to the 
Examples section hereinbelow. 
It has been found that the mild conditions which can be preferably utilized 
for covalently coupling hyaluronidase to SYNPOL do not affect the 
antigenic epitopes of hyaluronidase in a significant manner; accordingly, 
a highly immunogenic compound is obtained. Solid-phase enzyme-linked 
immunoassays (ELISA) have shown that anti-hyaluronidase antibodies can 
recognize the epitopes of hyaluronidase which have been conjugated to 
SYNPOL, demonstrating that these epitopes are retained. 
It has been found also that the enzymatic site of hyaluronidase is retained 
after the covalent coupling of hyaluronidase to SYNPOL. In particular, it 
has been observed that the substrate degradation rate of hyaluronidase 
alone is substantially identical to the substrate degradation rate of 
hyaluronidase which has been covalently coupled to SYNPOL. Furthermore, 
hyaluronidase when coupled to SYNPOL, is significantly more stable than 
the native enzyme. This has been demonstrated using hyaluronidase enzyme 
inactivation tests, including thermostability trials and resistance to 
heparin mediated inhibition. 
The enhanced stability of hyaluronidase provided by its conjugation to 
SYNPOL provides a broad spectrum of other utilities for the compound of 
the present invention. In addition to its ability to inhibit helminth 
infections, it is anticipated that the compound of the present invention 
may be used to elicit an immune response to other pathogens or organisms, 
for example, those pathogens or organism which make use of hyaluronidase 
to digest tissue, such as, for example, certain bacteria and their toxins. 
In addition, the compound of the present invention can be used to block 
the action or to localize the spreading of venoms containing hyaluronidase 
(such as bee and snake venoms). 
The present invention also includes within its scope methods of using 
hyaluronidase covalently coupled to SYNPOL to treat fibrosis in 
vertebrates by administering a composition comprising hyaluronidase 
covalently bound to SYNPOL. 
In addition, the compound of the present invention can be used in 
cosmetology as the active ingredient in creams and other products used to 
make skin smoother and more tender. In this regard, it should be noted 
that materials containing human sperm have been used as skin-care products 
in Russia. However, the use of such a product is highly limited because of 
the instability of the hyaluronidase. Since the compound of the present 
invention is stable, soluble, and non-toxic, it has immediate applications 
in this area, and indeed investigations of it uses in this regard have 
been carried out. The present invention also includes within its scope the 
use of hyaluronidase covalently bound to SYNPOL as a spreading factor to 
increase the efficacy of medications. It can also be used to improve 
diffusion and hasten absorption in medical use, for example, as an 
ingredient in an antibiotic solution for the treatment of bovine mastitis 
in veterinary use. In the past, unstabilized hyaluronidase has been used 
in these contexts (cf. The Merck Index, 8th Edition for example), and 
accordingly, the stabilized form of hyaluronidase provided by the present 
invention is expected to provide significant advantages over compositions 
which use the native form of hyaluronidase. 
The present invention also includes within its scope the use of 
hyaluronidase covalently bound to SYNPOL in the following therapeutic 
contexts where free (unstabilized) hyaluronidase has been shown to have 
beneficial effects: myocardial infarctions (cf. E. J. Flint et al, The 
Lancet, Apr. 17 (1982) pp.871-874 and also D. Maclean et al, Science, vol. 
194, pp.199-200 (1976)); improving retinal function (cf. B. S. Winkler et 
al, Arch. Opthalmol. 103 (1985) pp.1743-1746); in combination with 
cytostatics in the treatment of cancer tumors (G. Baumgartner et al., J. 
Exp. Clin. Cancer. Res. 4 (1985) p.3, and W. Scheithauer et al., 
Anticancer Res., vol 8, pp.391-395 (1988)); in the management of 
tuberculous spinal arachnoiditis (cf. M. Gourie-Devi et al., J. Neurol. 
Sci., vol. 102, pp.105-111 (1991)); for the management of encapsulated 
brain abscesses in high-risk risk patients (cf. A, Pasaoglu, Acta 
Neurochir., vol. 100, pp.79-83 (1989)). Furthermore, hyaluronidase has 
been used in vitro for depolymerizing hyaluronic acid in a cell free 
system, for instance, or for stimulating hyaluronic acid synthetase in eg. 
cell-culturing procedures (L. H. Philipson et al, Biochemistry 24 (1985) 
pp.7899-7906). The present invention also includes within its scope the 
use of the compound comprising hyaluronidase covalently bound to SYNPOL in 
these contexts. 
In addition, the invention includes within its scope SYNPOL-antigen 
conjugate vaccines in which the antigen comprises proteins similar to 
hyaluronidase, for example, vaccines containing as the antigen other 
enzymes used by pathogens to digest/degrade tissue (including collagenases 
and proteinases of different specificities). 
The invention also includes within its scope the use of SYNPOL coupled with 
an allergen in order to abolish allergic reactions within a host to a 
given allergen. Extensive investigation has shown that administering such 
an antigen-SYNPOL conjugate induces preferentially the production of 
non-allergic antibody isotypes against the allergen in question. These 
normal non-pathogenic isotypes compete with the previously existing 
allergenic one (e.g. IgE immunoglobulins) and specifically abolish the 
allergy. This method of specific desensitization has been clearly 
demonstrated and is now in the first stage of clinical trials. 
The invention also includes within its scope the use of SYNPOL coupled with 
the following antigens to enhance the immunogenicity of the coupled 
antigens, thereby serving to promote the induction of an effective 
prophylactic immune response: beta-subunit of cholera toxin, hemagglutinin 
from envelope of types A and B influenza viruses, p. 90 toxin from B. 
anthracis, the Vi antigen from salmonella, porin protein from the cell 
wall of E. coli and salmonellae, synthetic fragments of the gp160 
env-protein of HIV-1, F(ab)2 fragments of immunoglobulins (in order to 
induce an anti-idiotype response). 
EXAMPLES 
The first two examples are illustrative of the preparation of two species 
of SYNPOL, as identified in the examples. 
Example 1 
Preparation of SYNPOL 1000-20/50 
A three step procedure was used to synthesize a copolymer of 
ethylenepiperazine N-oxide and N-acetylethylenpiperazinium bromide. 
(1) The initial polymer, 1,4-ethylenepiperazine, was synthesized in the 
first step. For this purpose, the living chain polymerization of 
1,4-diazabicyclo2.2.2!octane was performed according to the following 
protocol. 
10 g of the preliminarily sublimed monomer and 0.05 g of ammonium bromide 
were sealed in 10 ml glass ampule. A vacuum of residual pressure 
5.times.10.sup.-3 mm Hg was produced in the ampule using a vacuum pump. 
The ampule was exposed for 25 hours at 200.degree. C. in a thermostat. 
Polymer yield was about 100%, M.W. 120,000 (estimated by LALLS-low angle 
laser light scattering). 
(2) The second step was performed to produce the N-oxide of 
poly-1,4-ethylenepiperazine. 
5 g of poly-1,4-ethylenpiperazine (M.W. 120,000, n=1000) were dissolved in 
250 ml of 1% acetic acid solution. Then, 4 ml of 30% H.sub.2 O.sub.2 were 
added, and oxidation lasted for 36 hours. After ultrafiltration and 
lyophilization, the N-oxide of poly-1,4-ethylenepiperazine (M.W. 110,000, 
z=0.5n) was obtained. 
(3) The alkylation of the above poly-N-oxide was performed during the third 
step. 
Poly-1,4-ethylenepiperazine N-oxide produced during the second step was 
dissolved in 125 ml of methanol and 16.5 g of bromoacetic acid were added. 
The alkylation reaction was carried out for 10 hrs. at 25.degree. C. The 
solvent was evaporated in a vacuum and the deposit dissolved in water, 
dialyzed against water for 24 hrs. and dried using lyophilization. 
Finally, the copolymer of ethylenepiperazine N-oxide and 
N-acetylethylenepiperazinium bromide of the following formula was obtained 
(see FIG. 2). 
The yield was 95%. The oxidation ratio was estimated by the chromometric 
(or titanometric titration) method and by the ratio of integral 
intensities of PMR-spectrum bands in region 2.5-4.5 m.d. The chromometric 
or titanometric titration method refers to the method of quantitative 
determination of N-oxide groups reduced by salts of bivalent chrome or 
trivalent titanium (Brooks, R. T. and P. D. Sternglanz, Anal. Chem., 1959, 
v. 31, N4, p. 561-565). Oxidation ratio amounted to z=0.5n. Alkylation 
ratio was determined by IR-spectra (1735 cm band) and PNR-spectra (2.5-4.5 
m.d. region) and accounted q=0.2n. 
Example 2 
Preparation of SYNPOL 200-35/65 
A copolymer of ethylenepiperazine N-oxide and N-acetylethylenepiperazinium 
bromide with M.W. 25,000 (n=200, q=0.35n, z=0.65n) was synthesized using a 
three step procedure, similar to the one of Example 1. 
(1) In the first step, 10 g of the preliminarily sublimed monomer and 0.11 
g of ammonium bromide were sealed in a 10 ml glass ampule. Then a vacuum 
(5.times.10.sup.-3 mm Hg) was produced in the ampule by a vacuum pump, and 
the ampule was kept at 200.degree. C. for 15 hours. The yield of 
poly-1,4-ethylenepiperazine was about 100%, M.W. 80,000 (measured by 
LALLS). 
(2) In the second step, the N-oxidation of poly-1,4-ethylenepiperazine was 
carried out as follows. 
5 g of poly-1,4-ethylenepiperazine obtained in the first step were 
dissolved in 250 ml of 1% acetic acid solution. Then 4.6 ml of 30% H.sub.2 
O.sub.2 was added at 2.degree.-4.degree. C. using gentle agitation. The 
oxidation lasted for 48 hours. Then after ultrafilter cleaning and 
lyophilization, the N-oxide of poly-1,4-ethylenepiperazine (M.W. 50,000, 
z=0.65n) was recovered. 
(3) The quantity of poly-1,4-ethylenepiperazine N-oxide produced in the 
step (2) above was dissolved in 125 ml of methanol and then 16.5 g of 
bromoacetic acid were added. The reaction of alkylation was carried out at 
30.degree. C. for 24 hours. The solvent was evaporated in a vacuum and the 
resulting deposit obtained was dissolved in water, dialyzed for 24 hours 
against water, and lyophilized. There was produced a copolymer of 
ethylenepiperazine N-oxide and N-acetylethylenepiperazinium bromide having 
the following formula (see FIG. 3). 
The yield was 95%. The oxidation and alkylation ratio, both estimated as in 
Example 1, were z=0.65n and q=0.35n respectively. 
The next four examples are illustrative of the preparation of compounds 
within the scope of the present invention and comprising the reaction 
products of hyaluronidase and various species of an immunostimulating 
carrier, namely, SYNPOL of the type which are the subjects of Examples 1 
and 2 above. 
Example 3 
Preparation of the Covalent Conjugate of Hyaluronidase (HYA) with SYNPOL 
1000-20/50 
A two-step procedure using the azide method was performed in order to 
synthesize the conjugate of HYA with SYNPOL 1000-20/50. 
(1) The first step of the procedure was used to produce the hydrazide of 
Synpol 1000-20/50`. 
A copolymer of ethylenepiperazine N-oxide and N-ethyl 
acetyl!ethylenepiperazinium bromide (n=1000, q=0.20, z=0.5) was 
synthesized according to the method described in Example 1 above except 
for one change in the third step: ethyl ester of bromoacetic acid was used 
for alkylation instead of bromoacetic acid. 500 mg of the copolymer were 
dissolved in 25 ml of methanol. Then 0.2 ml of hydrazine hydrate (0.2 
mmol) was added and the reaction continued for 24 hours at 20.degree. C. 
After the methanol was evaporated, the reaction product was harvested and 
dissolved in water. Thereafter, ether extraction was performed and the 
main product isolated by ultrafiltration on hollow fibers (Amicon) and 
lyophilized. 
The content of hydrazide groups in the modified polymer was estimated using 
a conventional method for primary amino groups determination by 
2,4,6-trinitrobenzenesulfonic acid S. L. Snyder and P. Z. Sobooinsky, 
"Improved 2,4,6-trinitrobenzenesulfonic acid method for determination of 
amine," Anal. Biochem., 1975, v. 64, N1, p. 284-288!. 
(2) In the second step, the reaction of condensation of HYA with the 
hydrazide of SYNPOL 1000-20/50 was performed in order to produce the 
covalent protein-polymer conjugate. 
To achieve this, 100 mg of the hydrazide of SYNPOL 100-20/50 were dissolved 
in 4 ml of 1 M HCl. The solution was stirred and cooled down to 
0.degree.-2.degree. C., and at the same time 1.15 ml of 3% sodium nitrite 
solution (0.5 mmol) were added. In 15 minutes, the pH of the activated 
SYNPOL 1000-20/50 solution was adjusted to 8.5 using 2 M NaOH. Thereafter, 
a solution of 12 mg of HYA in 10 ml of 0.05 M phosphate buffer (pH 8.5, 
potassium dihydrogen phosphate, disodium hydrogen phosphate) was added to 
the aforementioned solution of activated SYNPOL 1000-20/50. The reaction 
mixture was stirred and cooled (2.degree.-4.degree. C.), and the pH was 
kept at 8.5 using 2 M NaOH during 12 hours reaction time. 
Gel-filtration on Biogel P-100 was used in order to fractionate the 
components of the reaction mixture and purify the HYA-synpol conjugate. 
The chromatography column (26.times.900 mm) was filled with Biogel P-100 
available from Biorad Inc. and equilibrated by 0.05 M phosphate buffer 
with 0.05 M NaCl (pH 7.5). Fractions were eluted using the same buffer and 
the output was controlled by a flow UV-photometer (226 nm). The conjugate 
that was obtained was subjected to fluorescence spectroscopy and 
polyacrylamide gel electrophoresis (PAGE) to estimate the protein content 
and to analyze the conjugate. It was shown that 1 mg of the conjugate 
preparation contained 0.10 mg of HYA. 
Example 4 
Preparation of the Covalent Conjugate of Hyaluronidase (HYA) with SYNPOL 
200-35/65 
A copolymer of ethylenepiperazine N-oxide and N-acetylethylenepiperazinium 
bromide (M.W. 25,000, n=200, q=0.35, z=0.65) was synthesized according to 
the method described in Example 2 above. The conjugation of HYA with the 
copolymer was performed as described in Example 3 above. The condensation 
of polymer with HYA was carried out using the polymer/protein ratio 5:1 at 
pH=8. The final preparation of conjugate contained 0.15 mg of HYA per 1 mg 
of conjugate. 
Example 5 
Conjugation of Hyaluronidase (HYA) to SYNPOL 1000-20/50 Using the Activated 
Ethers Method 
A copolymer of ethylenepiperazine N-oxide and N-acetylethylenepiperazinium 
bromide (n=1000; q=0.2n; z=0.5) was synthesized according to Example 1 
above. A two-step chemical procedure was used in order to get the covalent 
conjugate of HYA with the copolymer. 
(1) In the first step, a succinimide ether of the copolymer was prepared. 
For this purpose, 100 mg of the copolymer were suspended in 4 ml of 
dimethylformamide and during stirring, 77.2 mg (0.30 mmol) of 
dicyclohexidcarbodiimide and 36 mg (0.30 mmol) of N-hydroxysuccinimide 
were added. The reaction lasted 24 hours during which the reaction mixture 
was stirred and cooled (2.degree.-4.degree. C.). The reaction mixture was 
the washed with dioxane, ethyl ether and acetone several times and dried 
in vacuum drier-box. The absence of low molecular admixtures was shown by 
thin-layer chromatography on "Silufol" plates in n-butanol:water:acetic 
acid (4:1:1). Then the content of activated ether groups was estimated by 
the standard method (T. Miron and M. Wilchek, Anal. Biochem.. 1982, v. 
126, N2, pp. 433-435) (0.1M NH.sub.3 water solution at pH=8.5, 259 nm, 
extinction coefficient=9700 1/mol.times.cm). The molar extinction 
coefficient "epsilon" was calculated from the Lambert-Beer equation: 
EQU D=epsilon.times.C.times.L, 
where: 
D--the value of optical density; 
C--the concentration of the compound in the solution examined; and 
L--the optical path. 
The content of activated ether group was 9.times.10.sup.-4 mol per 1 g of 
the modified copolymer. 
(2) In the second step of the procedure, the covalent coupling of HYA to 
the above succinimide ether of the copolymer was carried out. For this 
purpose, 100 mg of the activated copolymer produced in step (1) above were 
dissolved in 10 ml of 0.05M phosphate buffer solution (pH 6.0), cooled, 
and during continual stirring, a solution of 15 mg HYA dissolved in 12 ml 
of 0.05M phosphate buffer (pH 7.5) was added. The reaction of condensation 
lasted 18 hours at 0.degree. C. Then the conjugate was isolated from the 
reaction mixture by column chromatography on the Biogel P-100 (BioRad) 
column and analyzed as described in Example 3 above. The final preparation 
of the protein-polymer conjugate contained 0.10 mg of HYA per 1 mg of 
conjugate. 
Example 6 
Preparation of the Covalent Conjugate of HYA to SYNPOL with a 2:1 Ratio in 
the Conjugate 
SYNPOL 1000-20/50 was synthesized according to the method described in 
Example 1 above. The conjugation of HYA with synpol was performed as 
described in Example 3 above with only one change in the procedure 
protocol: the initial ratio of SYNPOL to HYA in the reaction mixture was 
2:1. The final conjugate preparation contained 0.3 mg of HYA per 1 mg. 
Example 7 
Vaccine Preparation 
The vaccine prepared consisted of the HYA-SYNPOL conjugate and an 
additional amount of SYNPOL itself acting as an immunoadjuvant. 
SYNPOL 1000-20/50 was obtained as in Example 1. The conjugation of HYA with 
Synpol was carried out as in Example 3 using the initial polymer/protein 
ratio 1:1. Namely, 5 mg of HYA was conjugated using the hydrazide method 
with 5 mg of SYNPOL 1000-20/50. Then the water solution of 40 mg of Synpol 
1000-20/50 was added to the purified conjugate, mixed and lyophilized. The 
HYA content in the final complex preparation was analyzed as in Example 3 
and showed 0.1 mg of HYA per 1 mg of the preparation. 
Example 8 
Vaccine Complex Containing the Derivative of Muramyldipeptide as the 
Immunoadjuvant 
The vaccine complex prepared was composed of both the HYA-Synpol conjugate 
and the glycosaminyl derivative of muramyldipeptide, a known 
immunoadjuvant. 
SYNPOL 1000-20/50 was synthesized as in the Example 1. The covalent 
conjugate of HYA with Synpol was obtained as in Example 3 using the 
polymer/protein ratio 1:1. Namely, 5 mg of HYA was conjugated with 5 mg of 
SYNPOL 1000-20/50. then 10 mg of 
N-acetylglucosaminyl-N'-acetyl-muramyl-L-alanyl-D-isoglutamine (GMDP) was 
added and the complex mixture lyophilized. 
Example 9 
Vaccine Complex Containing Aluminium Hydroxide as an Immunoadjuvant 
The vaccine complex consisted of HYA-SYNPOL conjugate and aluminium 
hydroxide as an immunoadjuvant. 
SYNPOL 1000-20/50 was obtained as in Example 1. The polymer/protein ratio 
1:1 was used during the covalent conjugation of SYNPOL with HYA using the 
method described in Example 3. The conjugate preparation was thoroughly 
mixed with a suspension of aluminium hydroxide ex tempore, just prior to 
immunization of animals. 
Example 10 
Preclinical Safety Evaluation of H-Polyvac 
H-Polyvac is a polymer-antigen vaccine against migratory forms of 
helminths. The vaccine is a conjugate of hyaluronidase, HYA, with the 
polymer immunostimulant SYNPOL. The protein antigen HYA is common for many 
forms of larval helminths. SYNPOL was developed and thoroughly 
investigated by the applicants. The polymer was shown to be safe for the 
human organism in a dose of 0.25 mg/kg and therefore may be recommended 
both as a stand-alone immunostimulant and as an adjuvant and/or carrier 
for vaccines. The Committee on Immunobiological Drugs has given permission 
for the injection of SYNPOL as a compound of conjugate vaccines. The 
recommended dose for agricultural animals (sheep, calf) of H-Polyvac is 4 
mg/animal, containing 0.5 mg of HYA antigen. 
The purpose of this study was a toxicological assessment of the H-Polyvac 
vaccine to evaluate its safety. In order to increase the reliability of 
the data obtained the study was carried out on 3 species of animals (mice, 
rats and guinea pigs). Not only the influence of doses close to the 
vaccination dose was investigated, but in addition the effects of 
overdosing were investigated with doses 10-100 times higher than the 
vaccination dose. The usage of high doses in a toxicological study permits 
one to establish the target organs as well as to obtain the toxicological 
characteristics of the preparation used. Pathological changes, which are 
detected during injection of high doses, are not considered to be 
contraindications for clinical trials, but give valuable information 
concerning the limitations of the tested preparation. 
Before presenting the actual details of the trials and the data obtained, 
the following remarks are helpful when interpreting the results. As can be 
seen in Table 8 below, pneumonia, lung atelectases and lung abscesses 
occurred in 10-20% of rats in the control placebo group. It is a well 
known fact among experts in animal pathology, that most rats, mice and 
other laboratory animals kept in conditions standard in the 
animal-breeding facilities of large laboratories are not completely 
healthy. They in fact suffer from various pathologies, which usually 
cannot be established without pathomorphological examination of their 
inner tissues and organs. 
For example, latent infections of a bacterial etiology have been carefully 
investigated in laboratory animals (K. Benirschke, editor, Pathology of 
Laboratory Animals, N.Y. 1978). The frequency of lung diseases in control 
(clinically healthy) rats is rather high. According to G. Paget and P. 
Lemon more than 99% of control laboratory rats have a latent pathology in 
their lungs (Pathology of Laboratory Animals. Eds. W. Ribelin and J. 
McCoy, Springfield, 1965, pp. 382-405). Similarly, J. Nelson has shown 81% 
frequency of pneumonia in control laboratory rats (Pathology of Laboratory 
Rats and Mice. Eds. E. Cotchin and F. Roe. Oxford, 1967. p. 259). The 
pathomorphology of the so-called "latent chronic respiratory disease" in 
laboratory rats has been described by J. Innes et al (Am.J.Path., 1956, 
v.32, pp. 141-160; and in Pathology of Laboratory Rats and Mice. Eds. E. 
Cotchin and F. Roe, Oxford, 1967, pp. 229-259) as well as by J. Lindsey et 
al. (Disease of Laboratory Animals Complicating Biomedical Research, 
Chicago, 1971, pp. 675-716). 
According to J. Lindsey (op.cit), E. Venzon et al. (Philipp.J.Vet.Med., 
1979, v.18, pp. 117-124), and M. Van Zwieten et al. (Lab.Anim.Sci., 1980, 
v.30, pp. 215-221) the hidden pathological processes in lungs of rats are 
mainly induced by Mycoplasma pneumoniae, Pasteurella spiralis and/or mouse 
pneumonia virus. 
Extensive examination of clinically normal WAG, August and Wistar inbred 
rats, and of noninbred animals of the production colonies of the Russian 
Academy of Medical Sciences, showed that 40-96% of rats were affected with 
chronic respiratory diseases. The respiratory organs were partially or 
totally involved in chronic catarrhal or catarrhal-purulent inflammation 
of the upper respiratory tract, trachea, bronchi, as well as in the 
development of chronic focal interstitial pneumonia (E. Abdrashitova, 
Respiratory organs of rats bred in the production colonies, 
Bull.Acad.Med.Sci.Russ., 1993, N9, 81-85). Concerning chronic enteritis, 
we note that its precise etiology has not been established, but it is 
likely they are symptoms of hidden infectious diseases. Most likely in 
these cases it was a clinically hidden chronic inflammation induced by 
certain kinds of bacteria as above. 
It is thus clear that so called "normal" animals often suffer from hidden 
infections. It is a widely accepted opinion that this occurs because of 
non-optimal living conditions, commonly found in most vivariums. While 
this situation is not optimal as regards research work and animal trials 
of compounds, there are, however, some positive conclusions concerning the 
control animals in the trials: since the animals used were weakened 
because of "hidden" infections, the trial results show the complete safety 
of H-Polyvac (10.times. doses injected 10-times) not only for healthy 
animals, but even for weakened ones. Finally, it may be added that the 
trial results obtained on normally-infected laboratory animals are 
significantly closer to those obtained in real farm conditions. 
1. Materials and Methods of the Study 
The program of preclinical safety evaluation of H-Polyvac included an 
evaluation of acute toxicity in mice during intraperitoneal infusion, and 
of chronic toxicity during daily H-Polyvac injections continued for ten 
days in doses 10.times. higher than the vaccination dose; this was 
accompanied by peripheral blood analysis, liver and renal functional 
tests, examination of the cardiovascular system and pathomorphological 
analysis of changes in internal organs. In addition, investigations of 
local reactions to the injections as well as allergic, mutagenic, 
pyrogenetic and carcinogenic effects were carried out. 
A dose of 4 mg (0.5 mg of protein for the sheep with a weight of 10-15 kg) 
was taken as 1 dose of the vaccine. 
2. H-Polyvac Acute and Subacute Toxicity Evaluation 
The average lethal dose of H-Polyvac was established in acute experiment on 
mice with a baseline weight of 25 g. The animals were carefully selected 
according to their body weight, each varying from the baseline weight by 
less than 1 g., i.e. by less than than 5%. Each dose was tested on 6 
animals with a period of observation of 16 days; mortality checks were 
performed daily. 
At the end of the 2 weeks of observation, the animals were killed and their 
organs examined morphologically. After H-Polyvac samples were dissolved in 
a physiological solution, a 5% solution was prepared and then injected 
intraperitioneally in doses containing 3 g/kg, 1.5 g/kg and 0.75 g/kg of 
vaccine respectively. 
The average lethal dose was determined using the probit-analysis technique 
under Litchfield and Wilcockson, which is the most widely applicable and 
allows one to obtain relatively complete information. 
The data in Table 1 demonstrate that H-Polyvac LD-50 is 1.66+0.04 g/kg. 
3. Assessment of the Local Response to H-Polyvac Injection 
Intracutaneous injections. Experiments assessing the local reactions to 
H-Polyvac injections were performed according to techniques recommended by 
Directive No. 31 of USSR Ministry of Health, "Unification of 
Immunobiological Drug Control Techniques". 
5 guinea pigs were used in this experiment; physiological solution and 
H-Polyvac (800 mg in 0.1 ml), diluted to 1:10 and 1:100 were injected 
once, intracutaneously, to different regions in a volume of 0.1 ml, after 
removing hair. 
The observation period was 1 month. 
Conclusion: there were no visible signs of skin inflammation during the 
period of observation. 
4. H-Polyvac Subacute Toxicity Evaluation 
The experiment was carried out on 60 male Wistar rats, with a baseline 
weight of 270-320 g. Animals were allocated to 3 groups, each group 
containing 20 rats. The first group of animals received 0.4 mg/kg of 
H-Polyvac, the second group received 4 mg/kg of H-Polyvac while the other 
third group acted as a control group and received physiological solution. 
H-Polyvac was injected intramuscularly each day for 10 days. Some animals 
were killed immediately after the termination of H-Polyvac administration, 
and the others 4 weeks after termination physiological, biochemical, 
hematological and histological tests were performed as well as regular 
body-weight evaluation. The results were then evaluated by statistical 
methods. 
Results: There was no significant difference in the body-weight gain of 
experimental animals as compared to the controls during the entire period 
of observation (6 weeks), at the same time body weight gain in animals, 
receiving 4 mg/kg is lower than in animals receiving 0.4 mg/kg (FIG. 4). 
4.1 Liver and Renal Functional Tests 
Blood serum analyses were performed using F-901 Biochemical Analyzer 
(Finland) and diagnostic kits LAHEMA (USSR). The results are presented in 
Table 2. The table shows that there was some increase in ALT activity 
after both 0.4 mg/kg doses and 4 mg/kg doses (14.5 and 19%, respectively). 
The biochemical range of blood serum of experimental animals did not 
differ from that of the controls 4 weeks after terminating the study. 
Daily diuresis and diuretic speed were examined in order to evaluate renal 
function. In addition, glomerular filtration and channel reabsorption were 
tested. The results are presented in Tables 3 and 4. Analysis of the data 
obtained demonstrated that neither renal filter membrane-permeability, 
channel reabsorption nor glomerular filtration were affected by daily 
injections of H-Polyvac for 2 weeks. 
4.2 Hematological Analysis 
The red blood cell count and total leukocyte count using Goryaev Chamber as 
well as the haemoglobin concentration, hematocrit ratio, color index and 
corpuscular haemoglobin concentration, were measured in order to evaluate 
the status of peripheral blood. The color index was determined as: 
##EQU1## 
Corpuscular haemoglobin concentration was determined as: 
##EQU2## 
where Ht=hematocrit and HB=haemoglobin. 
The results are presented in Table 5. 
The data demonstrate, that the hematocrit ratio decreases significantly 
only in the group receiving the 10-fold dose of 4 mg/kg as compared to 
control group. This decrease does not exceed normal physiological 
variations of that parameter in rats. There were no other significant 
abnormalities in the peripheral blood, both immediately after 10 H-Polyvac 
injections and one month after terminating the administration. 
4.3 Central Nervous System (CNS) Status Examination 
The experiments were performed on male Wistar rats. H-Polyvac was injected 
intramuscularly each day for 10 days, while the control group received 
physiological solution. The evaluation of H-Polyvac's influence on the 
functional state of the CNS was carried out the day after final injection 
and then repeated one month later. 
The following tests were performed: 
orientation reactions and locomotion activity which are integrative 
parameters reflecting CNS status and neuro-muscular activity; 
"Hole" reflex, which also characterizes orientation reactions; 
spinal cord "tail flick" reflex, which characterizes animal pain 
perception; 
"string"-test, which characterizes muscle tone and movement coordination. 
These tests constitute a complete examination of the CNS status. 
(Methodical recommendations in use of behavioral reactions of animals in 
toxicology studies, Kiev, 1980.) 
The results are shown in Table 6 and 7. A decrease in locomotion activity 
(races) occurs in both groups, after termination of administration, while 
in the group receiving 0.4 mg/kg the difference from control is 
statistically significant (p&lt;0.05). Locomotion activity is similar to 
control after the convalescence period. There were no significant 
differences in other tests as compared to the control animals. 
4.4. Pathomorphological Analysis 
Pathomorphological analyses were performed on male Wistar rats. H-Polyvac 
was administered intramuscularly, in 10 injections, each one of which 
contained 0.4 mg/kg and 4 mg/kg respectively. Physiological solution was 
used as a control. Study material was taken twice: directly after the 
termination of H-Polyvac injections (first series, 26 animals), and one 
month after final administration (second series, convalescence period, 25 
rats). All animals were sacrificed by decapitation. 
Necropsy was carried out after blood collection for biochemistry analysis, 
then a macroscopic examination of internal organs, serosa, and cavities 
was performed as well as measurements of organ weights, color, 
bloodfilling level, hemodynamic disturbances or other abnormalities. 
For histological analysis the following organs, organ and tissue samples 
were taken from 36 animals (6 rats in each group): liver, kidneys, heart, 
lungs, testes, adrenal, thymus, spleen, lymph nodes of different 
localization, brain, spinal cord, stomach, intestine, colon, pancreas, 
thyroids, pituitary, subcutaneous cellular fat, and muscles in the place 
of injection. Whole organs and samples of organs and tissues were fixed in 
10% formaldehyde saline, washed, treated with ethanol and placed in 
paraffin. At least 2 sections were placed on pieces of glass and stained 
with hematoxylin-eosin. 
Pathological and reactive changes were frequently observed in the control 
animals' organs because of the dramatic spread of infections and parasite 
diseases (see above); hence, the frequency of abnormalities detected in 
each group. 
4.4.2. Results of Macroscopic Examination 
Changes observed during external examination and necropsy in animals, 
killed directly after the termination of H-Polyvac administration and 4 
weeks later are presented in Tables 8 and 9. Both color and bloodfilling 
levels of the experimental subject killed directly after the termination 
of H-Polyvac administration and also those killed 4 weeks later were the 
same as control parameters. Table 10 displays the parameters of absolute 
internal weights in rats of the first and second series of experiments. 
There was a nonsignificant decrease of spleen weight in the first series 
group receiving 0.4 mg/kg, while a nonsignificant increase of groin lymph 
node weight occurred in a group receiving 4 mg/kg of H-Polyvac. The 
relative index of the weight of inner organs of animals sacrificed 
directly after the termination of H-Polyvac administration as well as of 
those killed 4 weeks later were similar to those of the control animals 
(Table 11). The expression "Relative index of the weight of inner organs" 
as used in this section, refers it is referring to the average of the 
ratio for each animal of the weight of the particular inner organ divided 
by the body weight and multiplied by 100%. 
Macroscopic examination of the place of H-Polyvac injection (subcutaneous 
cellular fat, muscle) did not show any hemodynamic disturbances in animals 
killed directly after the termination of H-Polyvac administration (first 
series). Only 1 out of 9 control animals and one out of 8 in the group 
receiving 0.4 mg/kg had puncture hemorrhages in subcutaneous cellular fat. 
In the second series (convalescence period) puncture hemorrhages in 
subcutaneous cellular fat were observed in one rat out of 8 control 
animals. There were no signs of inflammation (redness, infiltrates) in all 
groups of the two series. 
4.4.3. Results of Microscopic Examination 
Place of injection. 
There were no abnormalities in the microcirculation system found during 
histopathological examination of subcutaneous cellular tissue and hip 
muscle in rats of the first series (control, 0.4 gm/kg and 4 mg/kg). There 
was no edema, subcutaneous infiltration and productive reaction in either 
experimental or control groups. 
Neither necrobiotic nor dystrophic changes in muscle fibers were observed 
in experimental and control animals. 
There were no signs of inflammation in muscle tissue of control rats. At 
the same time, moderate mononuclear infiltrates were observed in 3 rats 
out of 6, receiving H-Polyvac in a dose of 0.4 mg/kg, with a prevalence of 
monocytes and macrophages. There was an increase both in the number and 
size of mononuclear infiltrates in 4 rats out of 6, receiving 4 mg/kg of 
H-Polyvac. Cells were also subjected to changes, with a prevalence of 
lymphocytes and frequent detection of plasma cells. 
Small mononuclear infiltrates were observed only in one animal from the 
control group, and in one rat from the group receiving 0.4 mg/kg of 
H-Polyvac, 4 weeks after the termination of H-Polyvac administration 
during histological examinations of the injection sites; small mononuclear 
infiltrates were observed in muscle tissue. There were no microcirculation 
abnormalities, dystrophic or necrobiotic changes. 
Internal organs 
Termination of H-Polyvac injection. 
Heart 
Multiple small perivascular hemorrhages were observed in the myocardium of 
one out of 6 control rats and in one out of 6 receiving 0.4 mg/kg of 
H-Polyvac. There were no other changes in the microcirculation system in 
either experimental or control groups. 
There was no edema or interstitial swelling, nor was there any inflammatory 
infiltration or stromal myocardial productive action. No discoid 
disintegration of myofibrils or myocytolysis was observed. 
Hyperemia of vessels and capillaries, sometimes with hemorrhages was 
observed in the lungs of all groups. Atelectases (partial contracture of 
alveoli wall) as well as small focal emphysema was detected in some of the 
animals of both experimental and control groups. Poly- and mononuclear 
infiltrates were observed in the interstices of both groups. Moderate 
activity of bronchus-associated lymphoid tissue was detected. H-Polyvac 
did not cause an increase in congestion events in the microcirculation 
system in doses of 0.4 mg/kg or 4 mg/kg, nor were there any signs of 
dystrophic, necrobiotic or inflammatory processes. 
There were no changes in the microcirculation system, no dystrophic or 
necrobiotic changes in glomerulae and tubules of nephrons, nor were there 
signs of inflammatory reactions to doses of 0.4 mg/kg or 4 mg/kg. 
There were no abnormalities in the liver microcirculation system in any 
groups. The structure of experimental and control organs was similar. No 
dystrophic changes in hepatocytes were found. One control rat had a small 
necrotic focus, while there were no necrobiotic signs in either 
experimental or control groups. Inflammatory changes (proliferation and 
hypertrophy of reticuloendothelial cells, poly- and mononuclear 
infiltrates) did not occur in any test groups. 
There were no changes in the microcirculation system of the pancreas in all 
animals examined. Structural changes of the exocrine part (pancreatic 
acini) were not observed. There were no signs of dystrophic, necrobiotic 
and inflammatory processes after H-Polyvac injections in doses of 0.4 kg 
or 4 mg/kg. 
Esophagus and stomach. There were no changes in the microcirculation system 
after H-Polyvac injections in doses of 0.4 mg/kg or 4 mg/kg. There were no 
signs of esophagus epithelium damage, nor of any epithelium damage in the 
cardiac, fundic or pyloric stomach regions, neither were there any 
inflammatory reactions. 
Signs of chronic enteritis together with thickening, deformation, and 
sometimes attachment of villi to each other were observed in the small 
intestine of all control animals. Epithelial dystrophy, and desquamation 
accompanied by villi stromal infiltration were found. Profound changes 
with crypt destruction occurred in several parts of the small intestine. 
The expression of chronic enteritis varied from mild to severely atrophic. 
There were no changes in groups receiving 0.4 mg/kg or 4 mg/kg doses of 
H-Polyvac in comparison with control groups. 
Colon. There were no changes in the microcirculation system after H-Polyvac 
injections in doses of 0.4 mg/kg or 4 mg/kg, nor in control groups. Colon 
mucosae were found to be intact, smooth and without edema. There were no 
signs of villi and crypt epithelium damage, dystrophic changes or 
desquamation. Regenerative epithelial activity appeared to be high. There 
were no inflammatory infiltrates. 
There were no changes in the thymic microcirculation system of control 
animals. One rat out of 6, receiving 0.4 mg/kg of H-Polyvac had a 
hemorrhage in the cortical zone of the thymus. A increase in the 
permeability of the vascular wall with further hemorrhages were registered 
in 3 rats out of 6 receiving 4 mg/kg of H-Polyvac. This group appeared to 
have signs of accidental thymus involution (reflected in a decrease of the 
cortical zone area and an increase of the connective tissue area). 
Inflammatory reactions were not found in any groups tested. 
Spleen. There were no changes in the microcirculation system after 
H-Polyvac injections in doses of 0.4 mg/kg or 4 mg/kg, or in control 
groups. A moderate trend of white pulp area reduction, as well as a 
diminution of the germinal centers was noticed in experimental groups. 
Cytoarchitectonics of the white pulp (ratio of germinal centers, 
T-dependent and marginal areas) was not altered. The activity of 
functional zones in experimental groups did not differ from that of the 
control group: the number of immunoblasts and plasma cells remained 
constantly low, as did the mitotic figures and pyknosis. There were no 
signs of dystrophic, necrobiotic or inflammatory processes in either 
experimental or control groups. 
Mesenteric and groin lymph nodes. There were no changes in the 
microcirculation system in either experimental or control groups, nor were 
there signs of dystrophic, necrobiotic and inflammatory processes. 
Parameters of activity of three types of immunity (T-, B- and 
macrophagal), evaluated according the three-rank system, were similar both 
in experimental and control groups and were within normal ranges. 
Brain. There were no changes in the microcirculation system seen during 
histological examination of brain sections, either in the control groups 
or in those which received H-Polyvac injections in doses of 0.4 mg/kg or 4 
mg/kg. There were no changes in the vessels of the ventricle or meninges. 
No structural changes of cortical or other brain neurons along with 
proliferative glial reactions were observed. 
Spinal Cord. There were no changes in the microcirculation system after 
H-Polyvac injections in doses of 0.4 mg/kg or 4 mg/kg, or in control 
groups. No structural changes of anterior and posterior horns, glial 
cells, white matter tissue, or inflammatory reactions were found. 
Pituitary. There were no changes in the microcirculation system either in 
experimental or control groups. Pituitary cytoarchitectonics 
(adenohypophysis, pars intermedia, neurohypophysis) in rats receiving 
H-Polyvac injections in doses of 0.4 mg/kg or 4 mg/kg, were similar to 
those of the control animals. All types of hormone producing cells were 
present in the adenohypophysis epithelium and there were no signs of 
dystrophic, necrobiotic or inflammatory reactions in all groups tested. 
Thyroid. There were no changes in the system in all groups studied. 
Structural organization of the thyroid functional unit (follicles) in the 
experimental group did not differ from the control group. Follicles were 
equally filled with nonvacuolized colloid, while epimonoium cells had 
monolayer cubic structure. Thyrocytes and C-cells appeared to be without 
dystrophic signs, increased growth or necrosis. Inflammatory stromal 
reactions were not found either in controls or in rats receiving H-Polyvac 
injections in doses of 0.4 mg/kg or 4 mg/kg. 
Adrenal. There were no changes in the microcirculation system after 
H-Polyvac injections in doses of 0.4 mg/kg or 4 mg/kg, or in control 
groups. The ratio of functional areas (cortical and medulla zones) 
remained similar to control in both experimental groups. There were no 
signs of dystrophic or necrobiotic changes in secretory cells or 
inflammatory reactions in all groups tested. 
Testes. There were no changes in the microcirculation system either in 
experimental or control groups. Epididymal structure remained undamaged 
and there were no dystrophic or necrobiotic changes, no desquamation of 
spermatogenic epithelium, Sertoli cells, nor was there any 
aspermatogenesis in all groups studied. No pathological changes or 
interstitial inflammation occurred in Leydig cells. 
Thus, pathomorphological analysis demonstrated that: 
chronic intramuscular injection of H-Polyvac in doses of 0.4 mg/kg and 4 
mg/kg does not produce dystrophic and necrobiotic changes in any organs 
examined; 
there were nonsignificant abnormalities in the microcirculation system of 
the following organs: in the thymus of one rat (out of 6) receiving 0.4 
mg/kg doses of H-Polyvac and in three rats (out of 6) receiving 4 mg/kg 
doses of H-Polyvac; in the heart of one control rat (out of 6) and one 
experimental rat (out of 6), receiving 0.4 mg/kg of H-Polyvac; 
intramuscular injection of H-Polyvac did not affect the frequency and 
intensity of spontaneous enteritis; 
there was a trend towards a decrease in the parenchymal working areas 
(areas of thymic cortical zone and splenic white pulp) of thymus and 
spleen in both experimental groups; however, it is difficult to interpret 
the results obtained because of the activation of the immune system as a 
result of the development of spontaneous chronic enteritis. Convalescence 
period. 
There were no signs of microcirculation abnormalities, dystrophic or 
necrobiotic changes, nor inflammatory reactions in any organs (except in 
the small intestine as mentioned above) in all groups tested, during 
histological examinations performed 4 weeks after terminating the 
administration of H-Polyvac. 
Atelectases and small-focal emphysema (in 2 rats out of 6) were observed in 
lungs of experimental and control groups. 
Signs of spontaneous chronic enteritis were found in the small intestine of 
all examined groups. Epithelium dystrophy and desquamation as well as 
round cellular infiltration of villi stromae was found. The intensity of 
enteritis varied from mild to severe in all tested groups. 
Signs of age involution were found in thymus of all groups (control, 0.4 
mg/kg and 4 mg/kg), accompanied by very low cortical activity. 
Moderate activity of splenic functional areas was found, combined with a 
low intensity of plasma cell reaction inside red pulp and with occasional 
extramedullar hemopoietic focuses. 
Conclusions: The results of pathomorphological analysis, performed in 
animals receiving 10 intramuscular injections of H-Polyvac, demonstrate, 
that H-Polyvac does not produce any pathological changes in all tested 
organs in doses 0.4 mg/kg and 4 mg/kg. 
5. Experimental Testing of H-Polyvac for Allergenicity 
Previous experiments have demonstrated the absence of Synpol sensitization 
activity. The purpose of this experiment was to check H-Polyvac vaccine 
for allergenicity, as SYNPOL is here used in conjunction with a protein 
antigen. 
The experiment was performed on 40 guinea pigs, divided into 4 groups: 
first--control group, second--protein antigen, third--SYNPOL, and 
fourth--H-Polyvac group, 4 subcutaneous injections 2 mg/kg 1 time a week. 
Cutaneous drop tests were carried out with a 2% water protein and SYNPOL 
solution. 
The technique of histamine provocation, designed by P. L. Zeltser and V. N. 
Drozdov in 1980, for assessment of allergenic effects of enzymatic 
hydrolytic preparations, was used in order to detect sensitization 
reactions. The technique involves intraperitoneal and intracutaneous 
injection of the tested antigen in combination with 0.03 gm/kg histamine, 
followed by anaphylactic reaction for 1.5-2 hours. According to the 
technique, histamine acts as a vascular adjuvant for more rapid and 
distinct reflection of weak allergens' sensitization characteristics. The 
quantitative ratio and functional status of different lymphocyte 
populations, in double rosette-formation and mitogen-stimulated 
rosette-formation (MSRF) reactions (Table 12) modified previously for 
guinea pigs examination (Dudintsava et al., 1982) were evaluated in 10 
experimental and 30 control animals, in order to determine the influence 
of H-Polyvac on immune lymphocytes. 
The results showed only primary irritative effects of the 2% protein 
antigen solution, reflected in the vessels' dilation and hyperemia, in the 
first minutes following the placement of H-Polyvac. There were no 
reactions showing development of immediate or delayed sensitization. 
These experiments demonstrated that neither sensitization nor quantitative 
or functional abnormalities of lymphocytes would develop after treatment 
of human organism with polymer antigenic vaccine. Certain adverse effects 
on vessels, though, should be taken into consideration when establishing 
contraindications. 
6. Assessment of H-Polyvac's Ability to Induce Dominant Lethal Mutations 
The experiments were carried out, according to the "Recommendations on New 
Drug Mutagenic Properties Control", adopted by the then USSR Ministry of 
Health in 1981. 
The experiments were continued for 5 weeks during both pre- and postmeiotic 
stages of spermatogenesis. H-Polyvac was injected intraperitoneally in 
doses of 0.6 mg/mouse and 30 mg/mouse and followed by the placement of 
three virginal females with the males treated with H-Polyvac. After 18 
days female mice were subjected to necropsy and considered for the number 
of dead and alive. 
Conclusions: H-Polyvac did not induce lethal mutations in embryonic cells 
of mice. At the same time 30 mg/mouse (1.5 g/kg) is close to LD-50, equal 
to 1.66+0.04 g/kg, and caused death of males the first day after 
administration. 
7. Assessment of H-Polyvac for Carcinogenic Activity 
The assessment of H-Polyvac carcinogenic activity was carried out according 
to the recommendations of "The Committee on Carcinogenic Substances". 
The reason for the performance of this experiment was the fact that the 
polymeric compound SYNPOL was present in the vaccine. 
The experiment was performed on two species of animals: 400 female Wistar 
rats with a 150-180 g baseline weight and 40 mice (C57BI/6), sensitive to 
tumor development. 
0.5 ml of 0.1% H-Polyvac solution in physiological saline was injected 
intraperitoneally, 2 times a week, for 8 months, in 14-15 injection 
courses with two-week interval in between. The animals received 100 mg of 
a protein/kg as a whole dose. Physiological saline was administered 
intraperitoneally to the control animals. Observations were generally 
continued until the natural death of animals, while some rats (exhausted, 
ill, with tumors) were sacrificed, the size of the tumor was established 
and macroscopic pictures of inner organs taken following their fixation in 
10% formaldehyde solution. 
The experiment lasted 2 years and 1 month. Four rats with spontaneous 
tumors were found in the control group in different periods within nine 
months of the initiation of the experiment, while 3 were found in the 
experimental group. 
The first tumors appeared 0.5 years later in experimental rats than in the 
control group. The speed and size of tumor development were also 
significantly lower in experimental rats. To emphasize H-Polyvac's 
carcinogenic activity, another series of experiment was performed in 
C57BI/6 mice, in order to evaluate the effect of H-Polyvac on Luis 
carcinoma development in lungs. 
Mice were chosen carefully according to their body weights (16 g) and 
divided into 4 groups, 10 rats in each group. Epidermal Luis lung 
carcinoma was transferred to all animals, by the subcutaneous injection of 
1:1 tumor solution with 199 medium, in a volume of 0.5 ml. 
Group 1 was the control group, group 2 and 3 received H-Polyvac in dose of 
5 mg/kg intraperitoneally and subcutaneously after 48 hours, while group 4 
received 2.5 mg/kg of carrier subcutaneously, correspondingly to the 
quantity of carrier administered to mice in groups 2 and 3. The treatment 
continued each day for 5 days. 
The weight of the animals and tumor size were determined, a week after 
tumor was transferred, i.e. at the end of the fifth day of H-Polyvac 
administration. The mice were killed, their body weight measured, and 
their tumors measured and separated and their weights determined. The 
results are presented in Table 14. The table demonstrates that both 
H-Polyvac and the carrier used alone cause a reduction in tumor 
development of 43-28% (by weight). The size and weight of tumors are 
significantly lower in all groups, as compared to the control group. 
Presumably, the polymeric immunostimulator SYNPOL has antitumor activity. 
These properties are preserved and/or increased in the polymer-antigen 
conjugate (Table 14, columns 2, 4). 
8. Conclusion 
A complete preclinical safety evaluation study of H-Polyvac was carried out 
in the Laboratory of Drug-Diagnostic Forms of the Russian Ministry of 
Health. 
H-Polyvac is a conjugate of a protein antigen with the polymeric 
immunostimulator Synpol. H-Polyvac is recommended as a vaccine against 
migrating forms of helminths in a dose of 0.05 mg of protein/kg of the 
animal's weight, that corresponds to a two-fold 0.4 mg/kg intramuscular 
injection of H-Polyvac. 
The safety evaluation of H-Polyvac was performed according to the 
requirements of the Pharmacological Committee of the USSR Ministry of 
Health (Directive of 31.12.1983), as well as the requirements of "The 
Veterinary Pharmacological Committee" (1974). 
The experiments were carried out in various species of animals: mice (CBA 
and C57BI/6 lines) 220 animals; Wistar rats, 180 animals; guinea pigs, 85 
animals. 
The results demonstrate that H-Polyvac is a practically nontoxic substance 
(class 5 of danger, according to SOST 12.1.07-76) with LD-50 being 
1.66+0.4 g/kg, during intraperitoneal infusion. 
Chronic toxicity was tested during multiserial daily injections of 
H-Polyvac, in vaccination (0.4 mg/kg) and 10-fold (4 mg/kg) doses. 
According to hematological, physiological, biochemical, immunological 
analysis, there were no negative effects of H-Polyvac on animals' body 
weights, behavior, central nervous system, cardiovascular system, liver 
and renal function or blood. 
No pathological changes in any internal organs and tissues, were determined 
by pathomorphological analysis. 
The absence of irritative activity in the place of injection, as well as 
the absence of allergic, immunotoxic and mutagenic activities were also 
established. 
No carcinogenic activity, during prolonged administration of H-Polyvac (8 
months) and 2.2 year observation of animals was detected. 
Therefore, the results of H-Polyvac safety evaluation study demonstrate the 
safety of H-Polyvac as well as a wide therapeutic ratio area (more than 
400). A therapeutic dose of 4 mg/kg of H-Polyvac can be considered as 
absolutely safe. 
Example 11 
Experimental and Pasture Trials 
To evaluate the effectiveness of the vaccine embodiment of the present 
invention, a series of experiments and pasture trials were conducted in 
the former USSR. The term "H-Polyvac" as used in this Examples section 
refers to the vaccine composition of the present invention. 
1. Protection conferred by H-Polyvac against experimental challenge with 
Echinococcus Granulosus 
1.1. Introduction 
The life cycle of Echinococcus granulosus consists of successive stages, 
first in dogs and then in either sheep, pigs or human. Two different 
stage-specific forms of E.granulosus live as parasites in the species 
mentioned. A scolex tape-like form lives in the intestine of dogs; the 
last link of the tape, which contains about thousand eggs (oncospherae) of 
E.granulosus, separates from the helminth and is disseminated in the 
excrement of the host. Sheep, pigs or humans can be invaded orally by eggs 
of E.granulosus. The cysta (bladder) form of echinococci then causes 
damage to the liver and/or lungs of the above animal species or humans. In 
order to evaluate the effectiveness of the H-Polyvac vaccine against 
echinococcosis, both artificial challenges of animals by E.granulosus as 
well as spontaneous invasion were investigated. Dogs were artificially 
infected using protoscolices, whereas lambs and piglets were infected 
using oncospherae of E.granulosus. 
1.2 Protection of dogs against an experimental challenge by E.granulosus. 
Initially, one needs to determine whether there is a protective effect of 
H-Polyvac against E.granulosus, and estimate approximately the range of 
effective doses for dogs. 
In the first experiment, that which involved 24 three-month old dogs, the 
range between 0.5 mg and 50 mg of H-Polyvac was investigated. Three 
separate groups containing 6 dogs each were injected twice using either 
0.5 mg, 5 mg, or 50 mg of H-Polyvac intramuscularly. The booster 
immunization was made 21 days after the first one. The remaining 6 dogs of 
a placebo group received injections of 0.9% NaCl saline. Two weeks after 
the booster immunization each of the 24 dogs was infected orally by 16,000 
protoscolices of E.granulosus. One month later all the dogs used were 
killed and dissected, and the number of E.granulosus helminths 
parasitizing the intestine obtained. 
The results represented in Table 15 clearly suggest that preliminary 
immunization of dogs significantly increases their resistance to an 
invasion by E.granulosus. The number of echinococci found in the intestine 
of dogs immunized by H-Polyvac, was 15-120 times less than in that of the 
placebo control group. Moreover, the protection intensity obviously 
depended on the H-Polyvac dose. The range about 5 mg. of H-Polyvac seemed 
to be optimal for immunizing dogs by twicerepeated injections. 
The goals of the next experiment were both a repetition of the measurement 
of the protective effects of H-Polyvac against echinococcosis, and an 
investigation of which H-polyvac doses near 5 mg are optimal. For these 
purposes, four separate groups of 3 dogs each, totalling 12 dogs, were 
used this time (Table 16). The group I dogs received 4 mg (i/m) of 
H-Polyvac twice with a 21 days interval between primary and secondary 
immunizations. The dogs of group II received 8 mg of H-Polyvac twice. The 
group III dogs were primed by 4 mg and then boosted by 8 mg of H-Polyvac. 
Dogs in the group IV received saline and served as a placebo control. Two 
weeks after the booster immunization, all the dogs used were infected 
artificially by oral administration of 5,000 protoscolices of 
E.granulosus. One month later, the animals were killed, dissected, and the 
resulting invasion intensity was established by counting the number of 
E.granulosus in the intestine. 
The results obtained are presented in Table 16, and confirm the data of the 
previous experiment. They demonstrate firstly the high efficacy of 
H-Polyvac in protecting dogs from intensive invasion by E.granulosus, and 
secondly that the 5 to 10 mg dose of H-Polyvac is the optimal one for 
intramuscular immunization of dogs. In fact, the control dogs of the group 
IV which did not receive H-Polyvac, were heavily invaded, possessing about 
2,000 echinococci in their intestine. On the contrary, the dogs of group I 
which were injected twice with 4 mg of H-Polyvac, possessed 100-300 times 
less echinococci. An increase of the H-Polyvac doses to 8 mg led to an 
enhancement of its efficacy in protecting against echinococci. In the 
intestine of dogs in groups II and III which were immunized by H-Polyvac 
using (8 mg+8 mg) or (4 mg+8 mg) schedules respectively, only a small 
number (from 0 to 3) of echinococci were found (Table 16). 
1.3. H-Polyvac against experimental challenge of pigs by E.granulosus. 
In total, 40 piglets, aged 1 month, were divided into 4 groups of 10 
piglets in each. All the animals in groups I, II and III were injected 
twice with 5 mg of H-Polyvac. The booster injection was made 7 days (group 
I), 14 days (group II), or 21 days (group III) after the priming injection 
of H-Polyvac. The ten piglets of the group IV received saline instead of 
H-Polyvac. 20 days after the booster immunization, all the animals were 
artificially challenged by E.granulosus using doses of 10,000 oncospherae 
per os. Seven months later, the pigs were killed and the bladder form of 
echinococci in the liver were counted. 
All the pigs of the placebo group IV underwent echinococci invasion. Large 
echinococci bladders of size 15-20 mm were found, and the number of 
helminths varied between 8 and 12 bubbles per liver (Table 17). On the 
contrary, no echinococci were found in most of the pigs which received 
H-Polyvac. Among the pigs of groups I, II and III, 70%, 90% and 80% of the 
animals respectively were absolutely free of echinococci. Furthermore, 
when helminths were found, their number varied between 1 to 4 echinococci 
bladders per liver. In addition, the helminth larvae found in the pigs 
immunized by H-Polyvac were very small in size (about 2-3 mm). 
Undoubtedly, the data shown in Table 17 demonstrates that of the range of 
intervals between primary and secondary injections of H-Polyvac shown, the 
interval of 14-21 days for immunization of piglets is preferred. 
The data obtained clearly shows the high efficacy of H-Polyvac regarding 
the prophylaxis of experimentally induced echinococcosis in pigs. In 
addition, the results mentioned above established both the dose and 
immunizing schedule for the effective vaccination of piglets by H-Polyvac. 
1.4. Protection of sheep against an experimental challenge by E.granulosus. 
Lambs aged 2-5 months were used in these investigations. Three separate 
experiments involving 12, 20 and 20 lambs respectivley were performed. 
The initial experiment was designed to test two doses of H-Polyvac in 
sheep, namely 5 mg and 10 mg, that were found as the effective doses for 
dogs and piglets. 12 lambs, each 3-4 months old, were divided into 3 
groups of 4 animals each (Table 18). The group I lambs were immunized 
using intramuscular injections by 5 mg H-Polyvac twice with a 21 day 
interval between priming and boosting injections. In the same manner, 10 
mg of H-Polyvac was administered twice to group II. The remaining 4 lambs 
served as a placebo control: they received 0.9% NaCl saline. 
Two weeks after the secondary immunization, all the lambs used were 
infected by E.granulosus by oral administration of 10,000 oncospherae. 
Four hundred days later, sheep of all three groups were killed, dissected, 
and the number of the bladder form of E.granulosus in their liver and 
lungs counted. 
The results obtained are represented in Table 18, in which the intensive 
invasion of the sheep in the placebo group is clearly noticeable. A large 
number of hydatid cysts of E.granulosus (mean=88 per animal) were found in 
the inner organs of sheep in this group. The animals that were immunized 
by H-Polyvac possessed 8-15 times less echinococci in their inner organs. 
Furthermore, the size of the echinococci bladders found in the H-Polyvac 
immunized sheep, was 1-2.5 mm, in contrast to 4-9 mm of that in the 
nonimmunized control animals. 
A subsequent similar experiment involved 20 lambs, 3 months old, divided 
into 3 groups. Eight lambs were immunized twice with the interval of 21 
days by 5 mg H-Polyvac intramuscularly (i/m) (Table 19). The other group 
of 8 lambs were injected subcutaneously (s/c) with a 5 mg dose of 
H-Polyvac, twice with a 21 day interval. Finally, the remaining 4 lambs 
received 0.9% NaCl saline, serving as a placebo control. Two weeks after 
the booster immunization, all the animals were infected by 10,000 
oncospherae of E.granulosus. Results of this experiment represented in 
Table 19 are very similar to those of the initial experiment (see Table 
18). 
In addition to the high efficacy of the immunization (performed by twice 
repeated injections of 5 mg of H-Polyvac) in protecting the lambs from 
experimental challenge by 10,000 oncospherae, the results of Table 19 show 
that both i/m and s/c modes of injection of H-Polyvac are acceptable for 
the vaccination procedure. 
In the third conclusive experiment, 20 lambs, each 4.5 months old, were 
divided into 3 groups according to the protocol represented in Table 20. 
Two different lots of H-Polyvac were used for immunization. The group I of 
8 lambs was immunized twice by 5 mg of the lot No. 1 of H-Polyvac. The 
group II of 8 lambs received 5 mg of the lot No. 2 of H-Polyvac. The 
remaining 4 lambs served as a placebo control. Later on, 2 weeks after the 
booster immunization, all 20 lambs were infected by E.granulosus by oral 
administration of 10,000 oncospherae. 
On the 425th day after the challenge, all the animals were slaughtered and 
dissected, and the number of the helminths in their liver and lungs 
counted. Results obtained reaffirm the high efficacy of H-Polyvac in 
protecting sheep against experimental invasion by a large number of 
oncospherae of E.granulosus (Table 20). In fact, control sheep of the 
placebo group were heavily invaded carrying a large number (mean=65 
helminths per animal) of echinococci in their liver and lungs. Both lot 
No. 1 and lot No. 2 of H-Polyvac, injected twice in a dose of 5 mg (i/m), 
strongly elevated the resistance of lambs to the challenge by 
E.granulosus. Some of those animals (3 out of 8 sheep in group I, i.e. 
37.5%) were totally free from echinococci in their inner organs. The 
remaining immunized sheep carried a small number (mean values are 3 and 
2.5 per animal in the groups I and II, respectively) of the echinococci 
bladders in their liver and lungs. Moreover, the helminth hydatid cysts 
found int he H-Polyvac immunized sheep were smaller in size (2-3 mm) than 
those found in the control animals (5-7 mm). 
1.5. Conclusions concerning the efficacy of H-Polyvac in preventing 
experimental echinococcosis in dogs, pigs and sheep. 
The investigations reviewed above in items 1.1-1.4 of H-Polyvac's activity 
in animals artificially infected using large doses of E.granulosus permit 
us to draw the following conclusions: (a) preliminary immunization of 
dogs, sheep, and pigs leads to a significant increase in their resistance 
to invasion by E.granulosus; (b) immunization by H-Polyvac defends the 
animal species mentioned even against a strong and intensive attack by 
echinococci, mimicked by an artificial acute challenge of animals with 
thousands of invasive helminths; (c) the protective effect of H-Polyvac 
against E.granulosus strictly depends on the H-Polyvac dose. Doses of 5-10 
mg of H-Polyvac injected twice, intramuscularly or subcutaneously, with a 
14-21 day interval between priming and boosting, were found to be optimal 
for immunizing either young dogs, 1 month old piglets, or 3-5 month old 
lambs. 
2. Protection conferred by H-Polyvac against experimental challenge with 
Dictyocaulus filaria 
For the hyaluronidase-antigen, included in H-Polyvac which is common for 
different species of helminths, one expects a protective action of 
H-Polyvac not only against echinococci, but also against other helminth 
species. This section is dedicated to the investigations made in order to 
estimate the protective effects of H-Polyvac in sheep as regards their 
resistance to an artificial challenge by a large number of D.filaria. This 
helminth infects animals as a invasive stage 3 larva (L3), entering an 
organism per os. The parasite then penetrates the intestine wall and 
migrates in the host, finally reaching its lungs. Here it grows for 
several weeks and develops into the mature stage. Growing within the lung 
tissue, the mature lungworms of 3-10 cm in size destroy their 
microenvironment in such a way as to result pneumonia, atelectases and 
abscesses in the lungs. Taking into consideration the life cycle of 
D.filaria, in the experimental challenge lambs were infected orally by 500 
L3-larvae and then 2 or 3 months later the number of D.filaria in the 
lungs was counted. 
In total, three separate experiments were performed using H-Polyvac in 
artificially induced dictyocaulasis. In the first experiment, 12 lambs, 
3-4 months old, were used: four of them were injected twice with 5 mg 
H-Polyvac (lot No. 1) intramuscularly, while the other 4 lambs received 
the same dose of lot No. 2 H-Polyvac. The booster immunization was made 21 
days after the primary one. The remaining four lambs served as a placebo 
control (Table 21). 
Two weeks after the booster immunization the animals were artificially 
challenged using doses of 500 dictyocaulae larvae per os. Two months 
later, the animals were killed and the number of D.filaria per organism 
counted. The immunization of lambs by H-Polyvac made them resistant to the 
intensive acute invasion by D.filaria (oral administration of 500 larvae). 
Two months after the challenge, about 15% of the immunized animals were 
totally free from D.filaria. The remaining H-Polyvac immunized animals 
were invaded by a small number (mean about 4 or 5 helminths per body), 
whereas the nonimmunized animals in the corresponding control group 
underwent heavy dictyocaulae invasion (mean value=67 helminths per 
animal). 
The following two experiments, summarized in the Table 22, produced very 
similar results to those of the Table 21. Briefly, the immunization of 2-3 
month old lambs using 5 mg (i/m, twice with 21 day interval) H-Polyvac 
protected animals against intensive challenge by 500 larvae of D.filaria. 
Thus, the protective influence of H-Polyvac is not restricted to 
E.granulosus. As was clearly shown in this section, H-Polyvac is also 
effective in the experimental model of an intensive invasion of lambs by 
D.filaria. The same H-Polyvac doses and immunization scheme were found to 
be effective in both helminthiases investigated. 
3. Protection conferred by H-Polyvac against experimental challenge with 
Fasciola hepatica 
Lambs 3-4 months old were used in the two experiments discussed below. The 
animals were immunized by H-Polyvac using 3-10 mg doses according to the 
protocols shown in Tables 23 and 24. Two weeks after the booster 
injection, the immunized, as well as control (placebo) animals, were 
invaded artificially by F.hepatica. For this purpose, the oral 
administration of 50 or 100 invasive larvae, named metacercaria, were 
used. Five to seven months later, the animals were killed and dissected, 
and the number of fasciolae in their livers were counted. 
The results obtained demonstrate a significant protective influence of 
H-Polyvac. The twice-repeated injection of 3 or 5 mg of H-Polyvac led to a 
50% decrease of the number of helminths surviving the host organism, even 
after an intensive challenge such as by oral administration of 100 
metacercariae (see Table 23). 
As soon as the challenge dose of metacercariae was halved, the protective 
effect of H-Polyvac reached 95-96% (see Table 24). 
The experiments performed constitute clear evidence that H-Polyvac induces 
an immune defense against artificial invasion by a large number of 
fasciolae. Taken together with the data reviewed above in items 1 and 2, 
they show a polyspecific protective effect of H-Polyvac against three 
different helminth species, namely, F.hepatica, D.filaria and 
E.granulosus. Moreover, if the H-Polyvac immunization protects animals 
from an intensive acute challenge by large numbers of helminths used, one 
definitely expects a protective effect of the vaccine in animals which 
undergo spontaneous invasion by helminths under natural conditions. The 
pasture trials of H-Polyvac showed these expectations to be correct. 
4. Polyspecific prophylaxis using H-Polyvac against spontaneous invasion by 
E.granulosus, D.filaria and F.hepatica 
It is a well-known fact that certain helminth invasions are characteristic 
of animal farms and/or animal farming regions. Some localities (or farms) 
are unfavorable as regards dictyocaulasis, other as regards larval 
cestodae invasions, fascioliasis, and so on. The word "unfavorable" here 
means that from year to year each new generation of animals born in the 
farm/region undergoes the same invasion and the percentage of animals 
subject to invasion is very high (often more than 50%), and finally that 
the number of helminths of the particular species parasitizing the animal 
organism is sufficiently high as to lead to the manifestation of clinical 
symptoms of helminthiasis in the animals. Taking this into consideration, 
the pasture trials of H-Polyvac were performed in different animal farms 
and regions, unfavorable as regards echinococcosis, dictyocaulasis or 
fascioliasis. Sometimes invasion by multiple helminth species (of those 
mentioned) occurred during the trials. 
4.1. Prevention of a spontaneous invasion of sheep and dog by E.granulosus 
under pasture condition. 
The initial trials were performed at the USSR state sheep farm, named 
"Koyadinsky" of the Karaganda District (Central Kazakhstan) and at the 
collective sheep farm "Leninsky Poot" of the Chadyr-Langoon region 
(Moldova). Both farms are unfavorable as regards echinococcosis. In fact, 
more than 60% of sheep at these farms were normally invaded by 
echinococci. 
According to the protocols shown in Tables 25 and 26, in total 75 2-3 month 
old lambs at the farm "Koyadinsky", as well as 135 lambs together with 11 
shepherd's dogs at the farm "Leninsky Poot", were used. The animals were 
immunized twice by 5 or 10 mg of either Lot No. 1 or Lot No. 2 of 
H-Polyvac. They were kept separately from the flock they belonged to 
during the period of immunization of H-Polyvac and two weeks after the 
booster immunization. They then joined their flocks and lived under normal 
pasture conditions, being in touch with animals invaded by echinococci. 
One year later at the farm "Koyadinsky", and 8 months later at the farm 
"Leninsky Poot", the sheep were slaughtered and the numbers of echinococci 
in their livers and lungs counted. As can be seen at the Tables 25 and 26, 
the control nonimmunized sheep were heavily invaded by echinococci. On the 
contrary, those which had been immunized using H-Polyvac were 
significantly more resistant to the invasion. Under the protection of 
H-Polyvac the percentage of invaded animals decreased from 78% to 26%, and 
the mean number of echinococci per animal invaded diminished from 25 to 2 
at the "Koyadinsky" or from 4.8 to 1.8 at the "Leninsky Poot". No 
significant differences between the protective effects of Lot No. 1 and 
Lot No. 2 of H-Polyvac were noticed. 
In addition to echinococci the invasion by the relative cestodae of other 
species, namely, Coenurus cerebralis and Cysticercus tenuicollis was found 
in animals slaughtered at the farm "Leninsky Poot". Data presented in 
Table 26 showed that H-Polyvac immunization substantially elevated the 
resistance of sheep not only to invasion by echinococci, but also to 
invasion by C.cerebralis and C.tenuicollis. 
The 11 shepherd's dogs were kept in pasture together with 135 sheep at the 
farm "Leninsky Poot". They were adult dogs and as they were already 
invaded by cestodae, they were treated by an antihelminthic named 
"Droncit" to get rid of their helminths before their immunization by 
H-Polyvac. 
Subsequently they were injected twice with 10 mg of H-Polyvac (i/m, 21 day 
interval) and lived together with flocks which they controlled as usual. 
Every month the excrements of all 11 dogs were tested to estimate whether 
or not dogs were reinvaded by cestodae. Finally, 8 months after the 
H-Polyvac immunizations the dogs received antihelminthic to verify the 
invasion by cestodae. During the entire period of observation no cestodae 
were found in the dogs which had received H-Polyvac. 
4.2. Protecting lambs from a spontaneous invasion of D.filaria. 
These pasture trials were performed at the collective sheep farm "Druzhba" 
(Bolshenarymsky Region, Eastern Kazakhstan) and at the sheep farm 
"Maximoka" (Anneny Noy Region, Moldova), both unfavorable as regards 
dictyocaulasis. Usually 90-100% of sheep at the farms "Druzhba" and 
"Maximoka" are invaded by dictyocaulae. In total, 220 and 68 lambs, 1.5-2 
months old, were used in trials at "Druzhba" and "Maximoka" respectively. 
The lambs were immunized by H-Polyvac and then two weeks after the booster 
immunization were sent to pasture and kept with the flocks to which they 
belonged. At the farm "Druzhba" the sheep were slaughtered 7 months after 
immunization, and the numbers of dictyocaulae in their lungs were 
ascertained (Table 27). The lambs involved in the trials at the farm 
"Maximoka" were not killed, but 5 months after the immunization by 
H-Polyvac the extent of D.filaria invasion was ascertained by coprological 
analysis of dictyocaulae larvae in their excrement (Table 28). 
4.3. Prophylaxis of spontaneous invasion of sheep by F.hepatica under 
pasture conditions. 
These trials were performed on the sheep farm "Poot Rybaka" (Dagestan, 
Russian Federation) and on the sheep farm attached to the Stavropol 
Station for Veterinary Research (Stavropol District, Russian Federation). 
In total, 243 and 50 lambs at the farms in Dagestan and Stavropol 
respectively were involved in the trials. Moreover, three different lots 
of H-Polyvac, namely Lots Nos. IG-4, IG-8 and IG-16, were used on three 
separate flocks of the sheep farm in Dagestan. Two lots of H-Polyvac (No. 
1 and No. 2) were used within the same flock at the farm in Stavropol. 
The lambs were immunized with H-Polyvac and 2 weeks later sent to join 
their flocks to live under normal pasture conditions. After 6, 7 or 10 
months the lambs were slaughtered, and the number of fasciolae in their 
livers was calculated. Data, represented at Tables 29 and 30, show that 
H-Polyvac immunization significantly diminishes the susceptibiltiy of 
lambs to invasion by fasciolae. 
All the lots of H-Polyvac used were efficacious in the prophylaxis of 
fascioliasis in lambs. It was useful to know that the combination of 5 mg 
H-Polyvac with 20 mg Synpol as an additional immunoadjuvant showed a 
slightly higher protective effect than 5 mg H-Polyvac itself. Later on 
this observation was confirmed and utilized during large scale trials of 
H-Polyvac (see item 5.2 below). 
Thus, though the characteristics of the infection by acute artificial 
challenge using a large amount of invasive helminths (E.granulosus, 
D.filaria, F.hepatica) were significantly different in comparison with 
those of the spontaneous challenge under normal pasture conditions, both 
experimental and pasture trials of H-polyvac showed the same high efficacy 
of the preparation in the prophylaxis of the above-mentioned 
helminthiases. 
5. State Trials of H-Polyvac 
After reviewing the data of the experimental and pasture trials given 
above, the State Chief Directorate for Veterinary Medicine and State 
Veterinary Inspection of the former USSR decided to perform large scale 
trials of H-Polyvac under pasture conditions (order No. 46 of 11 May 
1990). The state control trial-design pursued at least two goals: firstly, 
the verification of the efficacy of H-Polyvac on animal farms situated in 
different geographical and climatic regions of the country, and secondly, 
an estimation of the protective effect of H-Polyvac using large 
populations of animals. 
5.1 The broad geography of the trials. 
The list of sheep farms, unfavorable as regards echinococcosis, 
dictyocaulasis, or fascioliasis, included farms located in Ukraine 
(Kharkov and Soomy Regions, Crimea District), Moldova (Anneny Noy Reg. and 
Garakly Region), Georgia, Uzbekistan (Samarkand Region), Central, South 
and Eastern Kazakhstan, southern parts of the Russian Federation (Dagestan 
and Stavropol Districts), and central parts of the Russian Federation 
(Nyzhny Novgorod, Voronezh, Belgorod and Belaya Tserkov). 
Table 31 summarizes the information about the localities and number of 
animals used during the State trials. The data from all the trials 
completely confirmed the initial results presented earlier. Briefly, it is 
convenient to summarize the results obtained using the efficacy 
coefficient (EC), that is: 
##EQU3## 
where C is the mean number of helminths per organism in the control group 
and V is the same parameter in the vaccinated group of animals having 
received H-Polyvac. 
Using the efficacy coefficient, the data show H-Polyvac's effectiveness 
ranged between 82% and 90% in the prophylaxis of dictyocaulasis, and about 
90% in the prophylaxis of fascioliasis, and finally was nearly 100% in the 
prevention of echinococcosis. As examples of the manner in which trials 
were conducted see below a brief discussion of the data from the trials 
involving thousands of sheep which were performed in Eastern Kazakhstan. 
5.2. Trials of H-Polyvac on large populations of sheep under pasture 
conditions. 
It is generally accepted opinion among experts in epidemiology and 
epizootiology, that the larger the population investigated is, the more 
precise the data obtained about the epidemiology of an infection. This is 
also true for estimating a new vaccine's effectiveness, and thus the data 
of H-Polyvac trials performed on thousands of lambs is of great value. 
Some examples are given below. 
The protective properties of H-Polyvac were tested at the collective farm 
"Druzhba" (Eastern Kazakhstan) on 11000 lambs in pasture. The vaccine was 
administered twice in doses of 5 mg per animal to 20-30 day old lambs with 
an interval of 21 days, 45 days before the lambs were sent to pasture. A 
20 year later when 1127 lambs were slaughtered 3-4 coenures per animal 
were found in 6 lambs, while no dictyocaulae, fasciolae, or echinococci 
were found. The percentage of invaded animals among unvaccinated sheep 
varied between 80% and 100% in different flocks on the farm. 
The subsequent year H-Polyvac was tested at the same collective farm 
"Druzhba" on 11700 lambs in pasture. This time the vaccine was injected 
twice in a dose of 5 mg plus 20 mg of Synpol per animal into 20-30 day old 
lambs with an interval of 21 days, 45 days before they were sent to 
pasture. Seven months later, after the slaughter of 5000 lambs, 1-3 
dictyocaulae were found in 25 lambs and no fasciolae or echinococci were 
found. The extensiveness of invasion in the control (unvaccinated) flocks 
was 90-100%. 
Both examples of large scale trials of H-Polyvac clearly demonstrate its 
very high efficacy under real animal farming conditions. 
TABLE 1 
______________________________________ 
H-Polyvac Acute Toxicity Evaluation 
Dose of Vaccine 
g/kg No of Alive 
Dead/Alive 
% Of Mortality 
______________________________________ 
3 6 5/1 83.1 
1.5 6 3/3 50.0 
0.75 6 0/6 0 
______________________________________ 
TABLE 2 
__________________________________________________________________________ 
Biochemical Blood Serum Parameters of Male Rats During 
Intramuscular Injection of H-Polyvac 
H-Polyvac 
Parameter 
Units Control 0.4 mg/kg 
4 mg/kg 
__________________________________________________________________________ 
after 10 injections 
Liver Mass 4.15 .+-. 0.11 
3.87 .+-. 0.10 
3.85 .+-. 0.10 
quotient 
Tot. Protein 
g/l 36.59 .+-. 0.56 
36.34 .+-. 1.15 
35.07 .+-. 1.04 
Glucose mM/l 8.90 .+-. 0.24 
8.65 .+-. 7.28 
9.28 .+-. 0.27 
Cholesterol 
mM/l 45.88 .+-. 3.37 
78.90 .+-. 3.51 
53.17 .+-. 4.05 
Urea mM/l 6.58 .+-. 0.32 
6.44 .+-. 0.30 
7.17 .+-. 0.27 
Creatinine 
mcM/l 74.4 .+-. 3.11 
72.4 .+-. 4.37 
77.9 .+-. 2.66 
Chlorides 
mM/l 101.5 .+-. 0.84 
101.0 .+-. 0.72 
102.2 .+-. 1.43 
ALT U/l 62.9 .+-. 3.65 
72.0 .+-. 2.57* 
74.9 .+-. 3.97* 
AST U/l 391.1 .+-. 41.87 
386.5 .+-. 19.70 
398.4 .+-. 32.21 
AP U/l 863.9 .+-. 71.62 
825.3 .+-. 125.9 
837.1 .+-. 70.38 
4 weeks after terminating the administration 
Liver Mass 3.71 .+-. 0.18 
3.74 .+-. 0.12 
3.31 .+-. 0.09 
quotient 
Tot. Protein 
g/l 38.70 .+-. 1.28 
41.00 .+-. 0.76 
41.62 .+-. 1.49 
Glucose mM/l 8.76 .+-. 3.20 
8.16 .+-. 4.09 
8.73 .+-. 3.57 
Cholesterol 
mM/l 54.15 .+-. 5.39 
50.01 .+-. 3.21 
46.49 .+-. 5.73 
Urea mM/l 7.36 .+-. 0.24 
6.52 .+-. 0.36 
6.90 .+-. 0.39 
Creatinine 
mcM/l 98.38 .+-. 3.13 
91.33 .+-. 5.98 
102.48 .+-. 4.78 
Chlorides 
mM/l 99.1 .+-. 0.26 
100.0 .+-. 1.53 
100.3 .+-. 0.83 
ALT U/l 75.8 .+-. 7.90 
78.2 .+-. 6.08 
69.4 .+-. 4.26 
AST U/l 343.7 .+-. 21.47 
376.6 .+-. 28.51 
355.9 .+-. 18.50 
AP U/l 491.0 .+-. 35.9 
461.9 .+-. 33.4 
518.3 .+-. 31.3 
__________________________________________________________________________ 
TABLE 3 
__________________________________________________________________________ 
Evaluation of Renal Function During H-Polyvac Injection 
H-Polyvac 
Parameter 
Substrate 
Units 
Control 0.4 mg/kg 
4 mg/kg 
__________________________________________________________________________ 
Liver Mass 0.67 .+-. 0.02 
0.70 .+-. 0.02 
0.66 .+-. 0.01 
Quotient 
Diuresis ml 9.26 .+-. 0.72 
9.10 .+-. 0.71 
8.60 .+-. 0.72 
Diuretic 0.0064 .+-. 0.0005 
0.006 .+-. 0.00049 
Speed ml/mi 0.0063 .+-. 0.00049 
serum g/l 36.59 .+-. 0.66 
36.34 .+-. 1.15 
35.07 .+-. 1.04 
Protein 
urine g/l 6.16 .+-. 0.12 
6.20 .+-. 0.24 
6.80 .+-. 0.37 
urine g/24 h 
0.057 .+-. 0.004 
0.056 .+-. 0.005 
0.057 .+-. 0.003 
Urea serum mm/l 6.58 .+-. 0.32 
6.44 .+-. 0.30 
7.17 .+-. 0.27 
urine mm/l 893.7 .+-. 26.9 
886.9 .+-. 77.6 
941.0 .+-. 72.6 
urine mm/24 h 
8.19 .+-. 0.45 
7.99 .+-. 0.75 
7.97 .+-. 0.57 
clear.* 
ml/min 
0.87 .+-. 0.04 
0.82 .+-. 0.05 
0.78 .+-. 0.05 
Creatinine 
serum mcm/l 
74.4 .+-. 3.11 
72.4 .+-. 4.37 
77.9 .+-. 2.66 
urine mcm/l 
24025 .+-. 998 
22782 .+-. 1399 
23102 .+-. 1388 
urine mcm/24 
219.7 .+-. 12.1 
206.8 .+-. 21.7 
194.6 .+-. 11.1 
clear.* 
ml/mi 
2.07 .+-. 0.18 
1.92 .+-. 0.19 
1.77 .+-. 0.11 
Chlorides 
serum mm/l 101.5 .+-. 0.84 
101.0 .+-. 0.72 
102.2 .+-. 1.43 
urine mm/l 51.6 .+-. 7.2 
67.2 .+-. 8.16 
65.0 .+-. 7.8 
urine mm/24 h 
0.54 .+-. 0.07 
0.60 .+-. 0.07 
0.55 .+-. 0.08 
__________________________________________________________________________ 
*clear. clearance 
TABLE 4 
__________________________________________________________________________ 
Evaluation of Renal Function 1 Month After H-Polyvac Injection 
H-Polyvac 
Parameter 
Substrate 
Units 
Control 0.4 mg/kg 
4 mg/kg 
__________________________________________________________________________ 
Liver Mass 0.68 .+-. 0.02 
0.69 .+-. 0.01 
0.67 .+-. 0.03 
Quotient 
Dieresis ml 11.3 .+-. 1.5 
11.0 .+-. 0.6 
10.9 .+-. 0.5 
Diuretic 0.0078 .+-. 0.001 0.0076 .+-. 0.0005 
Speed ml/min 0.0076 .+-. 0.0004 
serum g/l 38.70 .+-. 1.28 
41.00 .+-. 0.76 
41.62 .+-. 1.49 
Protein 
urine g/l 5.48 .+-. 0.39 
5.66 .+-. 0.28 
5.30 .+-. 0.53 
urine g/24 h 
0.0062 .+-. 0.007 
0.062 .+-. 0.003 
0.057 .+-. 0.005 
Urea serum mm/l 7.38 .+-. 0.24 
6.52 .+-. 0.36 
6.90 .+-. 0.39 
urine mm/l 806.8 .+-. 49.9 
852.0 .+-. 90.3 
859.2 .+-. 64.8 
urine mm/24 h 
9.20 .+-. 1.05 
9.40 .+-. 0.98 
9.50 .+-. 0.73 
clear.* 
ml/min 
0.85 .+-. 0.09 
0.96 .+-. 0.09 
0.98 .+-. 0.10 
Creatinine 
serum mcm/l 
98.38 .+-. 3.13 
91.33 .+-. 5.98 
102.48 .+-. 4.78 
urine mcm/l 
21637 .+-. 217 
23871 .+-. 241 
20748 .+-. 139.5 
urine mcm/24 
245.5 .+-. 20.6 
259.5 .+-. 30.5 
226.1 .+-. 22.9 
clear.* 
ml/mi 
1.70 .+-. 0.24 
1.98 .+-. 0.22 
1.59 .+-. 0.20 
Chlorides 
serum mm/l 99.1 .+-. 0.26 
100.0 .+-. 1.53 
100.63 .+-. 0.83 
urine mm/l 61.3 .+-. 8.3 
53.8 .+-. 2.9 
67.8 .+-. 4.12 
urine mm/24 h 
0.68 .+-. 0.08 
0.60 .+-. 0.03 
0.75 .+-. 0.073 
__________________________________________________________________________ 
*clear. clearance 
TABLE 5 
__________________________________________________________________________ 
Peripheral Blood Analysis in Male Rats After 10 Days of 
Intramuscular Injections of H-Polyvac 
H-Polyvac 
Parameter 
Units 
Control 0.4 mg/kg 
4 mg/kg 
__________________________________________________________________________ 
Leukocytes 
10/l 
13.75 .+-. 0.94 
12.19 .+-. 0.99 
12.84 .+-. 1.22 
(11.7 .div. 17.0) 
(9.44 .div. 15.6) 
(9.0 .div. 15.5) 
Erythrocytes 
10/l 
5.39 .+-. 0.57 
4.84 .+-. 0.12 
5.03 .+-. 0.14 
(3.75 .div. 7.00) 
(4.60 .div. 5.15) 
(4.65 .div. 5.50) 
Haematocrit 
47.56 .+-. 0.60 
49.50 .+-. 1.07 
44.67 .+-. 1.11 
(p &lt; 0.05) 
Ratio % (45 .div. 51) 
(44 .div. 53) 
(40 .div. 50) 
Haemoglobin 
g/l 204.3 .+-. 3.0 
206.6 .+-. 2.4 
198.6 .+-. 5.1 
(186 .div. 217) 
(198 .div. 217) 
(166 .div. 213) 
Colour 1.17 .+-. 0.12 
1.27 .+-. 0.04 
1.21 .+-. 0.04 
Parameter (0.90 .div. 1.58) 
(1.18 .div. 1.38) 
(1.08 .div. 1.30) 
Corpuscular 
43.02 .+-. 0.87 
41.87 .+-. 0.99 
44.54 .+-. 1.06 
Hb Concent- 
% (38.82 .div. 46.30) 
(38.65 .div. 48.18) 
(40.00 .div. 48.50) 
ration 
__________________________________________________________________________ 
TABLE 6 
______________________________________ 
CNS Functional Status Parameters in Male Rats after 
10 Day Intramuscular Injections of H-Polyvac 
H-Polyvac 
Control 0.4 mg/kg 4 mg/kg 
Parameter N = 9 N = 8 N = 9 
______________________________________ 
Orientative- 
Explorative 
Behaviour (U) 
Races 72.8 .+-. 6.3 
43.0 .+-. 8.0 
(p &lt; 0.01) 
55.2 .+-. 7.5 
Sets 25.2 .+-. 4.7 
25.5 .+-. 7.5 26.0 .+-. 7.9 
reflex 3.1 .+-. 0.7 
2.0 .+-. 0.5 5.1 .+-. 0.8 
Pain Perception 
6.76 .+-. 0.42 
7.04 .+-. 0.42 6.57 .+-. 0.23 
Threshold (c) 
"String" 56.7 .+-. 3.0 
54.4 .+-. 4.1 51.1 .+-. 3.9 
Test (mm) 
______________________________________ 
TABLE 7 
______________________________________ 
CNS Functional Status Parameters in Male Rats One 
Month of Reconvalescence after Intramuscular 
Injections of H-Polyvac 
H-Polyvac 
Control 0.4 mg/kg 4 mg/kg 
Parameter N = 9 N = 8 N = 9 
______________________________________ 
Orientative- 
Explorative 
Behaviour (U) 
Races 40.6 .+-. 5.1 
40.6 .+-. 8.4 
33.9 .+-. 5.7 
Sets 14.6 .+-. 4.2 
28.4 .+-. 7.9 
18.7 .+-. 6.4 
reflex 3.7 .+-. 0.8 
2.6 .+-. 0.6 
3.6 .+-. 0.4 
Pain Perception 
6.71 .+-. 0.40 
6.23 .+-. 0.33 
7.54 .+-. 0.27 
Threshold (c) 
"String" 48.6 .+-. 3.5 
44.1 .+-. 2.4 
45.2 .+-. 2.8 
Test (mm) 
______________________________________ 
TABLE 8 
______________________________________ 
Frequency of Pathological Signs Observed in Animals, 
Killed Directly after the Termination of the Drug 
Administration 
H-Polyvac 
Control 0.4 mg/kg 4 mg/kg 
Changes N = 9 N = 8 N = 9 
______________________________________ 
Pneumonia 2 1 2 
Lung Lobe Athelectasis 
1 1 1 
Lung Abscess 1 1 2 
Punctual haemorrhages 
1 1 2 
in Thymus 
Punctual Haemorrhages 
3 5 5 
in Groin Lymph Node 
Punctual Haemorrhages 
0 1 2 
in Myocardium 
Punctual Haemorrhages 
1 1 0 
in the Place of 
Injection 
Puncutal Haemorrhages 
1 0 0 
in Liver 
______________________________________ 
TABLE 9 
______________________________________ 
Frequency of the Pathological Changes During Necropsy 
4 Weeks after Terminating the Administration of 
H-Polyvac 
H-Polyvac 
Control 0.4 mg/kg 4 mg/kg 
Changes N = 9 N = 8 N = 9 
______________________________________ 
Pneumonia 1 1 2 
Punctual Haemorrhages 
0 1 0 
in Lungs 
Punctual Haemorrhages 
1 2 2 
in Thymus 
Punctual Haemorrhages 
3 2 2 
in Groin Lymph Node 
Punctual Haemorrhages 
1 0 0 
in the place of 
Injection 
______________________________________ 
TABLE 10 
__________________________________________________________________________ 
Absolute Inner Organs Weight (in mg) of Animals, 
Receiving H-Polyvac 
Termination of administration 
H-Polyvac 
Group Control 0.4 mg/kg 4 mg/kg 
Organs N = 9 N = 8 N = 9 
__________________________________________________________________________ 
Liver 13.99 .+-. 0.49 
12.70 .+-. 0.65 
12.59 .+-. 0.53 
Kidneys 2.25 .+-. 0.08 
2.28 .+-. 0.07 
2.16 .+-. 0.05 
Heart 1.07 .+-. 0.04 
1.07 .+-. 0.4 
1.04 .+-. 0.03 
Lungs 1.63 .+-. 0.05 
1.57 .+-. 0.07 
1.53 .+-. 0.04 
Thymus 0.38 .+-. 0.04 
0.35 .+-. 0.05 
0.34 .+-. 0.03 
Spleen 1.81 .+-. 0.09 
1.53 .+-. 0.08* 
1.83 .+-. 0.09 
Adrenals 0.06 .+-. 0.002 
0.057 .+-. 0.002 
0.056 .+-. 0.002 
Testes 3.20 .+-. 0.09 
3.45 .+-. 0.09 
3.27 .+-. 0.10 
Groin Lymph Node 
0.08 .+-. 0.007 
0.07 .+-. 0.006 
0.09 .+-. 0.002 
Body weight (g) 
317.7 .+-. 11.2 
327.4 .+-. 10.7 
326.4 .+-. 7.9 
__________________________________________________________________________ 
Convalescence Period 
H-Polyvac 
Group Control 0.4 mg/kg 4 mg/kg 
Organs N = 8 N = 8 N = 9 
__________________________________________________________________________ 
Liver 13.69 .+-. 0.41 
13.87 .+-. 0.48 
12.00 .+-. 0.70 
Kidneys 2.54 .+-. 0.06 
2.57 .+-. 0.09 
2.43 .+-. 0.08 
Heart 1.15 .+-. 0.04 
1.12 .+-. 0.04 
1.17 .+-. 0.05 
Lungs 1.64 .+-. 0.05 
1.76 .+-. 0.08 
1.67 .+-. 0.06 
Thymus 0.25 .+-. 0.02 
0.24 .+-. 0.02 
0.24 .+-. 0.02 
Spleen 1.27 .+-. 0.08 
1.49 .+-. 0.09 
1.48 .+-. 0.13 
Adrenals 0.064 .+-. 0.002 
0.064 .+-. 0.001 
0.06 .+-. 0.001 
Testes 3.38 .+-. 0.11 
3.36 .+-. 0.12 
3.44 .+-. 0.09 
Groin Lymph Node 
0.048 .+-. 0.006 
0.063 .+-. 0.008 
0.071 .+-. 0.03* 
Body weight (g) 
370.3 .+-. 7.1 
373.0 .+-. 15.4 
367.8 .+-. 12.3 
__________________________________________________________________________ 
*Significant difference as compared to control (p &lt; 0.05) 
TABLE 11 
__________________________________________________________________________ 
Relative Internals Weight (in mg) of Animals, 
Receiving H-Polyvac 
Termination of Administration 
H-Polyvac 
Group Control 0.4 mg/kg 4 mg/kg 
Organs N = 9 N = 8 N = 9 
__________________________________________________________________________ 
Liver 4.16 .+-. 0.11 
3.87 .+-. 0.09 
3.85 .+-. 0.10 
Kidneys 0.67 .+-. 0.01 
0.70 .+-. 0.02 
0.69 .+-. 0.02 
Heart 0.32 .+-. 0.01 
0.33 .+-. 0.01 
0.32 .+-. 0.01 
Lungs 0.48 .+-. 0.02 
0.48 .+-. 0.02 
0.47 .+-. 0.02 
Thymus 0.11 .+-. 0.009 
0.11 .+-. 0.01 
0.11 .+-. 0.01 
Spleen 0.54 .+-. 0.03 
0.47 .+-. 0.02 
0.56 .+-. 0.02 
Adrenals 0.02 .+-. 0.0007 
0.02 .+-. 0.0008 
0.02 .+-. 0.0009 
Testes 0.96 .+-. 0.04 
1.06 .+-. 0.03 
1.00 .+-. 0.03 
Groin Lymph Node 
0.02 .+-. 0.002 
0.02 .+-. 0.002 
0.03 .+-. 0.001 
Body weight (g) 
337.7 .+-. 11.2 
327.4 .+-. 10.7 
326.4 .+-. 7.9 
__________________________________________________________________________ 
Convalescence Period 
H-Polyvac 
Group Control 0.4 mg/kg 4 mg/kg 
Organs N = 8 N = 8 N = 9 
__________________________________________________________________________ 
Liver 3.72 .+-. 0.16 
3.74 .+-. 0.11 
3.31 .+-. 0.12 
Kidneys 0.69 .+-. 0.02 
0.69 .+-. 0.02 
0.68 .+-. 0.03 
Heart 0.31 .+-. 0.008 
0.30 .+-. 0.11 
0.33 .+-. 0.008 
Lungs 0.44 .+-. 0.009 
0.47 .+-. 0.02 
0.46 .+-. 0.012 
Thymus 0.07 .+-. 0.007 
0.07 .+-. 0.006 
0.07 .+-. 0.016 
Spleen 0.34 .+-. 0.02 
0.40 .+-. 0.02 
0.42 .+-. 0.03 
Adrenals 0.02 .+-. 0.0005 
0.02 .+-. 0.0006 
0.02 .+-. 0.0005 
Testes 0.91 .+-. 0.02 
0.91 .+-. 0.03 
0.94 .+-. 0.03 
Groin Lymph Node 
0.011 .+-. 0.001 
0.02 .+-. 0.002 
0.02 .+-. 0.002 
Body weight (g) 
370.3 .+-. 7.1 
373.0 .+-. 15.4 
367.8 .+-. 12.4 
__________________________________________________________________________ 
*Significant difference as compared to control (p &lt; 0.05) 
TABLE 12 
__________________________________________________________________________ 
Quantitative Ratio and Functional State of Various 
Lymphocyte Populations after Treatment with 
H-Polyvac in Guinea Pigs 
Double Rosette-Forming 
Animal Reaction MSRF-Reaction 
Groups No of lymphocytes 
with Con A 
Doses No T B D O B D 
__________________________________________________________________________ 
2 mg/kg of 
10 49 .+-. 1.9 
7 .+-. 0.9 
5 .+-. 0.7 
39 .+-. 2.4 
1.0 .+-. 0.6 
2.2 .+-. 0.3 
H-Polyvac 
Intact 6 47 .+-. 3.5 
8 .+-. 1.2 
5 .+-. 0.9 
40 .+-. 4.6 
0.9 .+-. 0.07 
1.7 .+-. 0.1 
control 
1 mg/kg of 
8 56 .+-. 4.2 
5 .+-. 0.9 
4 .+-. 0.9 
31 .+-. 2.5 
0.9 .+-. 0.05 
2.1 .+-. 0.3 
H-Polyvac 
Intact 6 41 .+-. 3.7 
6 .+-. 1.4 
4 .+-. 0.9 
40 .+-. 4.6 
1.1 .+-. 0.14 
2.03 .+-. 0.3 
control 
__________________________________________________________________________ 
TABLE 13 
__________________________________________________________________________ 
Registration of Dominant Mutations in Embryonic Cells of Mice 
Stage of Level of 
Mutager 
Spermato- 
Substance 
No of 
Fertile 
Post- 
Mortality 
Mutagenic 
Activit 
genesis 
Infused 
Pregnant 
% implant 
Induced 
Effect 
Ratio 
__________________________________________________________________________ 
1 week 
H-Polyvac, 
29 96.6 2.3 0 0 0 
6 mg/mouse 
control 
28 93.3 2.0 0 0 0 
2 week 
H-Polyvac, 
29 96.6 2.0 0 0 0 
0.6 mg/mouse 
control 
29 96.6 4.0 0 0 0 
3 week 
H-Polyvac, 
30 100.0 
3.2 0 0 0 
0.6 mg/mouse 
control 
28 93.3 4.2 0 0 0 
4 week 
H-Polyvac, 
30 100.0 
0.3 0 0 0 
0.6 mg/mouse 
control 
30 100.0 
2.4 0 0 0 
5 week 
H-Polyvac, 
29 96.6 3.5 0 0 0 
0.6 mg/mouse 
control 
29 96.6 0.2 0 0 0 
__________________________________________________________________________ 
Note: 10 males were used both in experiment and control. 
TABLE 14 
__________________________________________________________________________ 
Influence of H-Polyvac and Polyoxidonium on the 
Development of Epidermal Luis Lungs Carcinoma in 
Mice C57BI/6 
Drug Method 
Days Tumour Tumour 
Dose of of No Of 
Volume 
% to Weight 
% to 
(mg/kg) Infusion 
Observation 
Dead 
(mm) Control 
(g) Control 
__________________________________________________________________________ 
Control s/c 14 30% 6080.25 
100 3.23 100 
H-Polyvac, 
s/c 14 10% 3621.75* 
59.6 1.85* 
57.0 
5 mg/kg 
H-Polyvac, 
i/p 14 10% 2869.82* 
47.2 2.33* 
72.2 
5 mg/kg 
Polyoxidonium, 
s/c 14 10% 3679.50* 
60.6 2.18* 
67.5 
2.5 mg/kg 
__________________________________________________________________________ 
*p &lt; 0.014 
TABLE 15 
__________________________________________________________________________ 
Immunization by H-Polyvac: 
Artificial 
Helminths in 
Interval Challenge by 
Intestine per 
Animals 
Priming 
(days) 
Boosting 
E. granulosus 
Dog (mean) 
__________________________________________________________________________ 
6 dogs 
0.5 mg 
21 0.5 mg 16,000 400 
i/m i/m protoscolices 
6 dogs 
5 mg 21 5 mg 16,000 50 
i/m i/m protoscolices 
6 dogs 
50 mg 21 50 mg 16,000 550 
i/m i/m protoscolices 
6 dogs 
saline 
21 saline 16,000 6,030 
protoscolices 
__________________________________________________________________________ 
TABLE 16 
__________________________________________________________________________ 
Helminths per 
Animals in Immunizing Scheme 
Animal in 1 Mo. 
Groups: by H-Polyvac: after Challenge 
Group Age Interval by 5,000 
No. Number 
(mo.) 
Priming 
(days) 
Boosting 
protoscolices 
__________________________________________________________________________ 
I 3 dogs 
3 4 mg 21 4 mg 8; 16; 12 
(i/m) (i/m) 
II 3 dogs 
3 8 mg 21 8 mg 2; 0; 1 
(i/m) (i/m) 
III 3 dogs 
3 4 mg 21 8 mg 3; 2; 2 
(i/m) (i/m) 
IV 3 dogs 
3 Saline 
21 Saline 
2475; 2500; 1573 
__________________________________________________________________________ 
TABLE 17 
__________________________________________________________________________ 
Immunization Scheme 
by H-Polyvac Helminth hydatid cysts 
Group 
Number of Intervals in Liver 
No. Animals 
Priming 
(days) 
Boosting 
EI* II** 
__________________________________________________________________________ 
I 10 piglets 
5 mg 7 5 mg 30% 3; 3; 4 
II 10 piglets 
5 mg 14 5 mg 10% 1 
III 10 piglets 
5 mg 21 5 mg 20% 1; 2 
IV 10 piglets 
Saline 
21 Saline 
100% 
10; 10; 9; 12; 8; 
12; 12; 11; 8; 9 
__________________________________________________________________________ 
*EI-extensiveness of invasion (percentage of invaded animals); 
**IIintensity of invasion (number of helminths per animal). 
TABLE 18 
__________________________________________________________________________ 
Helminths in Liver & Lungs 
400 days after Challenge 
Lambs Approx. Size 
in Group: 
Immunization by H-Polyvac: 
Number of 
of hydatid 
Age Interval Echinococci 
cysts 
Number 
(mo.) 
Priming 
(days) 
Boosting 
per Animal 
(mm) 
__________________________________________________________________________ 
4 3-4 5 mg 
21 5 mg 
3; 6; 4; 11 
1-2.5 
i/m i/m (mean = 6) 
4 3-4 10 mg 
21 10 mg 
9; 13; 8; 12 
1-2.5 
i/m i/m (mean = 11) 
4 3-4 Saline 
21 Saline 
52; 78; 77; 146 
4-9 
(mean = 88) 
__________________________________________________________________________ 
TABLE 19 
__________________________________________________________________________ 
Helminths in Liver 
& Lungs: 
Lambs Echinococci 
Size of 
in Group: 
Immunization by H-Polyvac: 
per hydatid 
Age Mode of 
Interval 
Animal cysts 
Number 
(mo.) 
Dose Injection 
(days) 
(mean + SD) 
(mm) 
__________________________________________________________________________ 
8 3 5 mg .times. 2 
i/m 21 6 + 4 1-3 
8 3 5 mg .times. 2 
s/c 21 5 + 4 1-4 
4 3 Saline 
s/c 21 63 + 23 
5-9 
__________________________________________________________________________ 
TABLE 20 
__________________________________________________________________________ 
Comparison of two different lots of H-Polyvac regarding their 
efficacy in protecting sheep from an experimental challenge 
by 10 000 oncospherae of E. granulosus. 
Helminth Bladders 
in Liver and Lungs 425 
days after challenge 
Sheep in Immunization by E. granulosus 
Group: by H-Polyvac: Number of 
Approx. Size 
Group Age Interval 
Helminths 
of hydatid 
No. Number 
(mo.) 
Lot Dose (days) 
per Animal 
cysts (mm) 
__________________________________________________________________________ 
I 8 4.5 Lot 5 mg .times. 2 
21 1; 0; 3; 8; 
2-3 
No. 1 
i/m 0; 0; 9; 4 
(mean = 3) 
II 8 4.5 Lot 5 mg .times. 2 
21 2; 2; 3; 2; 
2-3 
No. 2 
i/m 3; 2; 3; 3; 
(mean = 2.5) 
III 4 4.5 Saline 
i/m 21 49; 64; 81; 65 
5-7 
(mean = 65) 
__________________________________________________________________________ 
TABLE 21 
__________________________________________________________________________ 
Helminths in Lungs in 
Artificial 
2 mo. after Challenge 
Lambs in Immunization 
Challenge 
Number of 
Percentage 
Group: by H-Polyvac: 
by Helminths 
of Animals 
Age Lot Dose D. filaria 
per Animal 
Invaded by 
Number 
(mo.) 
No. (i/m) 
(per os) 
(mean + SD) 
Dictyocaulae 
__________________________________________________________________________ 
4 3-4 Lot 1 
5 mg .times. 2 
500 4 + 2 85% 
larvae 
4 3-4 Lot 2 
5 mg .times. 2 
500 5 + 1 87% 
larvae 
4 3-4 Saline 
2 ml 500 67 + 9 100% 
larvae 
__________________________________________________________________________ 
TABLE 22 
__________________________________________________________________________ 
D. filaria in Lungs in 
Lambs in Immunization 1-2 Months after Challenge: 
Group: by H-Polyvac Helminths 
Percentage 
Exp. Age Interval per Animal 
of Invaded 
No. Number 
(mo.) 
Priming 
(days) 
Boosting 
(mean) 
Animals 
__________________________________________________________________________ 
2 5 2.5-3 
Saline 
21 Saline 
30 100% 
18 2.5-3 
5 mg 21 5 mg 4 89% 
i/m i/m 
3 10 2-3 Saline 
21 Saline 
32 100% 
10 2-3 5 mg 21 5 mg 1 60% 
i/m i/m 
__________________________________________________________________________ 
TABLE 23 
__________________________________________________________________________ 
Fasciolae 
Lambs in per Liver 
Group: Immunization by H-Polyvac: 
Artificial 
5 mo. after 
Age Interval Challenge by 
the Challenge 
Number 
(mo.) 
Priming 
(days) 
Boosting 
F. hepatica 
(mean + SD) 
__________________________________________________________________________ 
4 3 3 mg 21 3 mg 100 12 + 4 
metacercariae 
4 3 5 mg 21 5 mg 100 10 + 3 
metacercariae 
4 3 Saline 
21 Saline 
100 20 + 8 
metacercariae 
__________________________________________________________________________ 
TABLE 24 
__________________________________________________________________________ 
Lambs in Artificial 
Fasciolae 
Group: Immunization by H-Polyvac: 
Challenge 
per Liver 
Age Priming 
Interval 
Boosting 
by 7 mo. after 
Number 
(mo.) 
(i/m) 
(days) 
(i/m) 
F. hepatica 
Challenge 
__________________________________________________________________________ 
10 3-4 5 mg 
21 5 mg 
50 1-3 
metacercariae 
10 3-4 10 mg 
21 10 mg 
50 1-2 
metacercariae 
10 3-4 Saline 
21 Saline 
50 28-37 
metacercariae 
__________________________________________________________________________ 
TABLE 25 
__________________________________________________________________________ 
The pasture trials of H-Polyvac at the sheep farm "Koyadinsky" 
Helminths in Liver 
& Lungs in 12 months 
after Immunization: 
Lambs in Number of 
Group: Immunization by H-Polyvac: 
Echinococci 
Age Priming 
Interval 
Boosting 
per Animal 
Approx. size 
Number 
(mo.) 
Lot (i/m) 
(days) 
(i/m) 
(mean + SD) 
(mm) 
__________________________________________________________________________ 
25 2-3 No. 1 
5 mg 21 5 mg 3 + 2 3-4 
25 2-3 No. 2 
5 mg 21 5 mg 2 + 1 2.5-3 
25 2-3 Saline 21 Saline 
25 + 10 
6-9 
__________________________________________________________________________ 
TABLE 26 
__________________________________________________________________________ 
The pasture trials of H-Polyvac at the sheep farm "Leninsky Poot" 
Lambs Cestodae in the Inner Organs in 
in Group: 
Immunization 
8 Months after the Immunization: 
Age by H-Polyvac: 
E. granulosus 
C. tenuicollis 
C. cerebralis 
Number 
(mo.) 
(i/m) EI* II** 
EI II EI II 
__________________________________________________________________________ 
45 2.5-3 
10 mg .times. 2 
26% 1.8 13% 2.5 0 0 
45 2.5-3 
5 mg .times. 2 
40% 3.1 46% 1.5 7% 1.0 
45 2.5-3 
Saline 78% 4.8 89% 4.0 11% 1.0 
__________________________________________________________________________ 
*EI-extensiveness of invasion; 
**IIintensity of invasion (see Table 3) 
TABLE 27 
__________________________________________________________________________ 
The pasture trials of H-Polyvac at the sheep farm "Druzhba" 
Helminths in Lungs in 7 mo. 
Lambs in Immunization after the Immunization: 
Group: by H-Polyvac D. filaria 
Age Priming 
Interval 
Boosting 
per Animal 
Percentage of 
Number 
(mo.) 
(i/m) 
(days) 
(i/m) 
(mean + SD) 
Animals Invaded 
__________________________________________________________________________ 
100 1.5-2 
5 mg 21 5 mg 2 + 1 12% 
(lot 1) (lot 1) 
100 1.5-2 
5 mg 21 5 mg 2 + 1 13% 
(lot 2) (lot 2) 
20 1.5-2 
Saline 
21 Saline 
13 + 3 100% 
__________________________________________________________________________ 
TABLE 28 
__________________________________________________________________________ 
The pasture trials of H-Polyvac at the sheep farm "Maximoka" 
Lambs in Period 
Percentage 
Group: Immunization by H-Polyvac: 
in of Animals 
Age Priming 
Interval 
Boosting 
Pasture 
Invaded by 
Number 
(mo.) 
(s/c) (days) 
(s/c) (mo.) D. filaria* 
__________________________________________________________________________ 
28 1.5-2 
5 mg 21 5 mg 6 25% 
25 1.5-2 
5 mg 21 10 mg 6 16% 
15 1.5-2 
Saline 
21 Saline 
6 100% 
__________________________________________________________________________ 
*Coprology 
TABLE 29 
__________________________________________________________________________ 
The pasture trials of H-Polyvac at the farm "Poot Rybaka" 
Lambs Immunization Period F. hepatica 
in Group: by H-Polyvac in per Liver 
Flock 
Number 
Age (mo.) 
Lot Dose (i/m) 
Pasture (mo.) 
(mean + SD) 
__________________________________________________________________________ 
A 40 2-3 IG4 5 mg .times. 2 
7 2 + 1 
40 2-3 (IG4 + PO) 
(5 mg + 20 mg) .times. 2 
7 0 
20 2-3 Saline 7 13 + 4 
B 13 3 IG-8 5 mg .times.p0 2 
7 4 + 3 
10 3 Saline 7 91 + 39 
C 100 2-2.5 
IG-16 5 mg .times. 2 
10 8 + 3 
20 2-2.5 
Saline 10 24 + 14 
__________________________________________________________________________ 
TABLE 30 
__________________________________________________________________________ 
The pasture trials of H-Polyvac at a sheep farm in Stavropol 
Lambs in Period 
Group: Immunization by H-Polyvac: 
in Number of 
Age Interval Pasture 
F. hepatica 
Number 
(mo.) 
Lot Priming 
(days) 
Boosting 
(mo.) 
per Liver 
__________________________________________________________________________ 
20 2-3 
No 1 
5 mg, i/m 
21 5 mg, i/m 
6 0 
20 2-3 
No 2 
5 mg, i/m 
21 5 mg, i/m 
6 0 
10 2-3 
-- Saline 
21 Saline 
6 7-32 
__________________________________________________________________________ 
______________________________________ 
Animals Helminthiasis 
Vaccinated Actual 
District by H-Polyvac in the Region 
______________________________________ 
Eastern Kazakhstan 
580 lambs Dictyocaulasis 
District 
Samarkand Reg., 
600 lambs Echinococcosis 
Uzbekistan 
Samarkand Reg., 
80 piglets Echinococcosis 
Uzbekistan 
Moldova 1000 lambs Echinococcosis 
Belgorod Reg., 70 lambs Fascioliasis 
Central Russ. Fed. 
Stavropol Reg, 500 lambs Fascioliasis 
South Russ. Fed. 
Dagestan, 120 lambs Fascioliasis 
South Russ. Fed. 
Nyzhny Novgorod Reg., 
50 lambs Fascioliasis 
Central Russ. Fed. 
Crimea Reg., 1000 lambs Dictyocaulasis 
Ukraine 
Georgia 50 lambs Fascioliasis 
Karaganda Reg., 
75 lambs Echinococcosis 
Central Kazakhstan 
Tseliograd Reg., 
30 lambs Dictyocaulasis 
Central Kazakhstan 
Dzhambul Reg., 500 lambs Echinococcosis 
South Kazakhstan 
Kharkov Reg., 50 lambs Dictyocaulasis 
Ukraine 
Soomy Reg., 100 lambs Dictyocaulasis 
Ukraine 
Eastern Kazakhstan 
7500 lambs Echinococcosis 
Eastern Kazakhstan 
11000 lambs Echinococcosis 
and 200 dogs Echinococcosis 
Eastern Kazakhstan 
1500 lambs Dictyocaulasis 
Eastern Kazakhstan 
11700 lambs Echinococcosis 
and 68 dogs Echinococcosis 
______________________________________