Oxidized viruses or viral antigens and utilization for diagnostic prophylactic and/or therapeutic applications

Novel and improved methods for diagnosis, prognosis, prophylaxis and therapy of viral infections are described. The novel methods employ a virus, viral antigen or fragment thereof in which "perturbation" of an oligosaccharide moiety renders the virus, viral antigen or fragment thereof more specifically recognizable or reactive with neutralizing antibody. As described, "perturbation" of an oligosaccharide moiety encompasses a variety of modifications such has one that (1) alters the chemical or physical structure of a carbohydrate residue that is naturally present; (2) that removes, wholly or in part, a carbohydrate residue; and/or (3) that prevents or alters addition of a carbohydrate residue. A variety of different methods for oligosaccharide "perturbation" are also described. In particular, the carbohydrate residue is altered by an oxidizing agent.

1. FIELD OF THE INVENTION 
The present invention relates generally to novel and improved methods for 
diagnosis, prophylaxis and therapy of viral infections. More particularly 
the invention relates to novel methods employing a virus, viral antigen or 
fragment thereof in which an oligosaccharide moiety is perturbed in such a 
way that the virus, viral antigen or fragment thereof is specifically 
recognized by or reacts specifically with neutralizing antibodies. The 
term "perturbed" oligosaccharide moiety is intended to encompass a variety 
of different types of modifications such as a modification (1) that alters 
the chemical or physical structure of a carbohydrate residue that is 
naturally present; (2) that removes, wholly or in part, a carbohydrate 
residue that is naturally present; and/or (3) that prevents or alters the 
addition of a carbohydrate residue (i.e. prevents or alters 
glycosylation). 
The perturbed viruses, viral antigens and fragments thereof prepared 
according to the present invention are useful for in vitro diagnostic and 
prognostic applications as well as for in vivo prophylactic and 
therapeutic applications. 
2. BACKGROUND OF THE INVENTION 
Viruses are important etiological agents of a wide variety of diseases. In 
animals the immune response comprises one of the basic mechanisms to fight 
viral infections. Classically, the immune response encompasses two facets: 
the B-lymphocyte antibody response, referred to as humoral immunity and a 
T-lymphocyte-mediated response, known as cell-mediated immunity. The 
present application is concerned particularly with the antibody response. 
While specific antibody of classes IgG, IgM and IgA can bind to any 
accessible epitope on a surface protein of a virion, only those antibodies 
which bind with reasonably high avidity to particular epitopes on a 
particular protein in the outer capsid or envelope are capable of 
neutralizing the infectivity of the virion. These are termed "neutralizing 
antibodies." "Neutralization" as used throughout the instant specification 
is intended to include not only (1) classical virus neutralization which 
results when antibody binds to a surface antigen of a virion which 
ordinarily binds to a receptor on the surface of a susceptible cell and 
thereby prevents infection of a susceptible cell or leads to opsonization 
but also includes (2) interactions such as the binding of an antibody to 
neuraminidase of influenza virus (IF) which results in inhibition of 
release of progeny virus particles from the plasma membrane of infected 
cells and slows virus spread and (3) binding of an antibody to fusion 
protein (F) of paramyxoviruses which does not prevent initiation of 
infection but does block the direct cell to cell spread of newly formed 
virions once infection has been established. 
Antibodies directed against irrelevant or inaccessible epitopes of surface 
proteins, or against internal proteins of the virion, or virus-coded 
non-structural proteins, such as virus-encoded enzymes can sometimes exert 
indirect immunopathological effects, but may play no role in elimination 
of the infection. These are "non-neutralizing antibodies." In fact, 
certain non-neutralizing antibodies not only form damaging circulating 
"immune complexes" but may actually impede access of neutralizing antibody 
and enhance the infectivity of the virion for some cells. For example, in 
the presence of sub-neutralizing concentration of neutralizing antibody or 
excess of non-neutralizing antibodies viruses such as togaviruses are 
actually taken up more efficiently by macrophages (via Fc receptors on the 
macrophage to which the virus-antibody complex binds). The virus multiples 
intracellulary to high titer inside the macrophages. Hence the 
non-neutralizing antibodies act as "enhancing antibody." Specific examples 
of such viruses include dengue virus types 1-4. 
Thus in response to a viral infection, two very different kinds of 
antibodies are produced: neutralizing antibodies and non-neutralizing 
antibodies. Each is present in the serum of infected individuals or 
individuals previously exposed to a virus or a viral antigen in varying 
amounts. In order to assess the true immunocompetent status of an 
individual it is necessary to know the absolute and relative amounts of 
both neutralizing and non-neutralizing antibodies. Yet conventional 
serological assays of antiviral antibodies do not, and in fact cannot, 
distinguish these two kinds of antibodies. Conventional serological assays 
measure the presence of both types of antibodies. Hence there is no 
serological method for measuring or assessing the true immune status or 
immunocompetence of individuals. 
The only conventional method for assessing virus neutralizing ability of 
serum of individuals has been the virus neutralization assay such as that 
described by Krech et al., Z. Immuno., Forsch. Bd. 141 S: 411-29 (1971). 
Neutralization assays require: (1) use of infectious virus and (2) cell 
culture techniques. Such assays are slow, cumbersome, labor intensive and 
expensive. Hence there has been a long-felt need for a rapid, inexpensive 
accurate serological method to assess the immunocompetent status of 
individuals. 
Examples of specific situations in which a rapid, easy test for assessing 
the immunocompetence of an individual is particularly important include, 
but are not limited to, the following. Firstly, exposure of a pregnant 
female to a virus such as rubella virus or cytomegolovirus poses 
significant risk of congenital defects for the fetus. Using conventional 
serological methods such as ELISA assays the titer of all IgM and IgG 
antibodies against the relevant virus, both neutralizing and 
non-neutralizing, may be determined. If the total IgM level is elevated 
indicating that the response is due to reaction by a presumably "naive" 
immune system, a therapeutic abortion will be recommended because it is 
unlikely that neutralizing antibodies against the virus are present. If, 
however, only the total IgG level is elevated, no therapeutic abortion 
will be recommended because the test cannot distingush whether the IgG's 
present are neutralizing or non-neutralizing antibodies. The patient is 
faced with a long stress-filled pregnancy which may end in a child with 
congenital defects. 
Secondly, exposure of (or reactivation of previous infection associated 
with immunosuppression) organ transplant or bone marrow transplant 
patients to viruses such as cytomegalovirus (CMV) poses significant risks 
of clinical disease states including pneumonia, hepatitis, retinitis, 
encephalitis, etc. Moreover, the glomerulopathy induced by CMV adversely 
affects the survival of kidney grafts, so that renal transplant patients 
face additional life-threatening organ rejection [see generally, White et 
al., eds., in Medical Virology, 3d ed., Academic Press, Inc., New York, 
pp. 419-426 (1986)]. 
Thirdly, viral infections pose significant, indeed often life-threatening 
risks for immuno-suppressed patients including cancer patients undergoing 
chemotherapy, and those afflicted with either congenital or acquired 
immunodeficiency such as acquired immune deficiency syndrome (AIDS). 
Fourthly, certain viral infections endemic to specific geographic areas 
pose significant risks, for example, for military or diplomatic personnel 
stationed in these areas. Specific examples include but are not limited to 
Rift Valley fever, dengue etc. Vaccines may protect by actively eliciting 
the production of neutralizing antibodies. Evaluation of the 
immunocompetent status of personnel to be sent to these areas following 
vaccination is important. 
In all the above examples, there is a need for rapid, serological methods 
for determining both the presence and titer of virus neutralizing 
antibodies. Examples of formats useful in such rapid, serological methods 
include but are not limited to Enzyme-Linked Immunosorbent Assays (ELISA), 
radioimmunoassays (RIA), immunofluoresence or other fluorescence-based 
assays, agglutination assays, etc. 
Hagenaars et al., J. Virol. Methods 6: 233-39 (1983) described a modified 
inhibition ELISA assay which showed some correlation between ELISA titers 
and neutralization assay titers for polio virus type I. Unlike the 
presently described assays, however, the modified inhibition ELISA of 
Hagenaars et al. was more complex and cumbersome. 
Dreesman et al., Virol. 69:700-09 (1976) investigated the site associated 
with hemagglutinating activity of adenovirus following oxidation of viral 
antigen and purified virus preparations. Animals immunized with oxidized 
preparations showed significantly decreased haemgglutination -- inhibiting 
antibody; however, neutralizing antibody titers were not substantially 
affected. In contrast, according to the present invention, mild oxidation 
of the oligosaccharide moiety of virus, viral antigen and virus fragments 
not only enhances the ability of the immunogen to elicit neutralizing 
antibody production, but more critically also elicits a protective immune 
response. 
3. SUMMARY OF THE INVENTION 
The present invention is based upon the surprising discovery that whole 
viruses, viral antigens and fragments thereof in which the structure of an 
oligosaccharide moiety of a viral glycoprotein has been "perturbed" are 
recognized more efficiently by serum, plasma or immunoglobulin fractions 
containing neutralizing antibody molecules. As used throughout the instant 
specification, the terms "perturbed" oligosaccharide and oligosaccharide 
"perturbation" are intended to encompass a variety of different types of 
modifications such as a modification (1) that alters the chemical or 
physical structure of a naturally occurring carbohydrate residue; (2) that 
removes, wholly or in part, a naturally occurring carbohydrate residue; 
and/or (3) that prevents or alters the addition of a carbohydrate residue 
to a virus, viral antigen of fragment thereof. Any techniques known in the 
art for achieving such oligosaccharide "perturbations" including, but not 
limited to chemical and enzymatic treatment and genetic engineering 
methods, are intended to fall within the scope of the present invention. 
Based on this discovery, one embodiment of the present invention provides a 
novel method for detecting, as well as quantitating, the presence of virus 
neutralizing antibodies in samples of body fluids such as serum, plasma, 
various immunoglobulin fractions, etc. The novel method of the invention 
has the following advantages over conventional assays for neutralizing 
antibodies: (1) does not require use of cell culture techniques; (2) does 
not require use of injectious virus; (3) comprises a straight-forward 
serological assay; and (4) is complete in 4 hours or less. In contrast, 
conventional assays for virus neutralizing antibodies require: (1) cells 
in culture; (2) infectious virus; (3) skilled personnel; and (4) 5 days or 
so before an answer can be obtained. Thus, the present method is faster, 
easier and less complex than conventional methods. It does not require 
skilled personnel trained in the handling of infectious viral materials 
and is safer for use because even trained personnel need not be exposed to 
infectious virus. 
Another embodiment of the present invention provides a novel method for 
preparing compositions comprising a virus, viral antigen or fragment 
thereof in which the oligosaccharide moiety is perturbed such that the 
compositions are useful for eliciting the formation of neutralizing 
antibodies which are capable of affording a protective immune response. 
Thus these compositions provide vaccine formulations which stimulate an 
active immune response for prophylaxis of viral infections. For example, 
according to this embodiment a viral antigen is prepared having a 
perturbed oligosaccharide moiety and administered as a vaccine formulation 
to actively elicit the production of protective antibodies. 
Another alternate embodiment of the present invention provides a variety of 
novel methods for preparing or identifying monoclonal or polyclonal 
neutralizing antibodies which can be administered to confer short-term 
passive immunity for prophylaxis and/or therapy of viral infections. For 
example, a perturbed viral antigen is used to identify those monoclonal 
antibodies prepared by hybridoma techniques which are capable of 
neutralizing virus and which are capable of affording a protective immune 
response. Such antibodies could be administered for prophylactic treatment 
of persons at risk of developing a particular viral disease.

5. DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to novel methods for diagnosis, prognosis, 
prophylaxis and therapy useful for a variety of viral infections. All the 
novel methods are based upon utilization of either whole virus, viral 
antigens or fragments of viral proteins in which "perturbation" of an 
oligosaccharide moiety renders the virus, viral antigen or fragment 
thereof more efficiently recognizable by neutralizing antibodies. 
5.1. OLIGOSACCHARIDE PERTURBATIONS 
According to the present invention, oligosaccharide "perturbation" 
encompasses a variety of modifications such as a modification (1) that 
alters the chemical or physical structure of a naturally occurring 
carbohydrate residue; (2) that removes, wholly or in part a naturally 
occurring carbohydrate residue; and/or (3) that prevents or alters the 
addition of a carbohydrate residue to a virus, viral antigen or fragment 
thereof. Illustrative examples of oligosaccharide perturbations include, 
but are not limited to the following. 
Whole viruses, viral antigens or fragments thereof are perturbed by mild 
oxidation of an oligosaccharide moiety of a viral glycoprotein. For 
example, chemical oxidation of the oligosaccharide moiety can be 
accomplished using a variety of oxidizing agents such as periodic acid, 
paraperiodic acid, sodium metaperiodate, and potassium metaperiodate. 
Oxidation using such oxidizing agents is carried out by known methods. For 
a general discussion, see Jackson, in Organic Reactions 2, p. 341 (1944); 
Bunton, in Oxidation Chemistry, Vol. 1., Wiberg, ed., p. 367, Academic 
Press, New York (1944). The amount of the oxidizing agent depends on the 
kind of virus or viral antigen, but generally is used in excess of the 
amount of oxidizable oligosaccharide. The optimal amount can be determined 
by routine experimentation. The optimal ranges include: pH from about 4 to 
8, a temperature range of about 0.degree. to 37.degree. C., and a reaction 
period of from about 15 minutes to 12 hours. During oxidation, light is 
preferably excluded from the reaction mixture in order to prevent over 
oxidation. Alternatively oxidation is achieved using an enzyme, such as 
galactose oxidase [Cooper et al., J. Biol. Chem. 234: 445-48 (1959)]. The 
influence of pH, substrate concentration, buffers and buffer concentration 
on the enzymatic oxidation are reported in Cooper et al., supra. 
Whole viruses, viral antigens, or fragments thereof are perturbed by 
culturing virus infected cells in the presence of glycosylation 
inhibitors. For example, infected cells may be cultured in the presence of 
glycosylation inhibitors including, but not limited to: tunicamycin, 
streptovirudins, and/or glycosidase inhibitors such as carbohydrate 
analogs such as 2-deoxy-D-glucose and the like, and castanospermine, 
norjirimycin, 1-deoxynorjirimycin, bromocenduritol, 1-deoxymannojirimycin, 
and swainsonine, etc. 
Whole viruses, viral antigens or fragments thereof are perturbed by 
enzymatically removing, either wholly or in part, a naturally occurring 
oligosaccharide moiety by exposing either virus infected cells in culture 
or isolated virus particles or fragments thereof to an enzyme which is 
specific for glycoside residues. Useful enzymes include neuraminidase, 
endo-beta-N-acetyglucosaminidases and the like. 
Perturbation of viral antigens according to the present invention is also 
accomplished using genetic engineering techniques. For example, a gene 
encoding a particular viral glycoprotein or fragment may be cloned and 
expressed in a bacterial organism. In such case, little to no 
glycosylation of the expressed viral protein occurs, resulting in a 
perturbed oligosaccharide. Alternatively, a gene encoding the desired 
viral glycoprotein or fragment can be cloned and expressed in a eukaryotic 
organism or cell culture. The eukaryotic organism or cell is then cultured 
in the presence of a glycosylation inhibitor such as tunicamycin or a 
glycosidase inhibitor such as castanospermine, norjirimycin, and the like 
resulting in expression of a protein or fragment having a perturbed 
oligosaccharide. Yet another alternative is to use site-selective 
mutagenesis techniques to alter a gene encoding a viral antigen or 
fragment in such a way as to remove or change the site(s) of 
glycosylation. 
In cases where the amino acid sequence of a viral glycoprotein or fragment 
is known, chemical methods of peptide synthesis provide yet another method 
for the preparation of a viral antigen or fragment thereof with a 
perturbed oligosaccharide. 
The foregoing are merely illustrative examples of methods of 
oligosaccharide perturbation. Any other techniques known to those of skill 
in the art are intended to be encompassed by the perturbed oligosaccharide 
moiety. 
The perturbed oligosaccharide of the virus, viral antigen or fragment 
thereof may be further modified, for example, by reduction with reducing 
agents such as sodium borohydride, cyanoborohydride and the like or by 
covalent attachment to a soluble or insoluble carrier or support. 
5.2. APPLICATIONS 
The viruses, viral antigens and fragments thereof according to the present 
invention are advantageously used for methods suited for a number of 
different applications. 
5.2.1. DIAGNOSTIC AND PROGNOSTIC APPLICATIONS 
According to one embodiment of the invention, the virus, viral antigen or 
fragment thereof having a perturbed oligosaccharide moiety is used in a 
variety of methods for assaying a sample of body fluids such as serum, 
plasma, cerebrospinal fluid, milk or partially purified immuno-globulin 
fractions or ascites fluid or supernatant produced by hybridoma cell lines 
or transformed cell lines which produce virus-specific antibody for the 
presence and titer of neutralizing antibodies. 
The viruses, viral antigens or fragments thereof are used as ligands 
(antigens) to detect the presence of virus neutralizing antibodies in 
assay systems including but not limited to systems such as: Enzyme-Linked 
Immunosorbent Assays (ELISA), radioimmunoassays (RIA), immunofluoresence 
or other fluorescence-based assays, agglutination assays, etc. Examples of 
viruses for which neutralizing antibody titers are assayed would include 
those such as described in Section 4.3. 
The titers obtained using assays with conventionally treated viruses, for 
example, conventional ELISA assays, which measure all antibodies, whether 
neutralizing or non-neutralizing, do not correlate with the titer 
determined in conventional virus neutralization assays. On the other hand, 
the titers obtained using the present viruses, viral antigens or fragments 
thereof having a perturbed oligosaccharide moiety (hereinafter referred to 
as a "perturbed antigen") have been found by Applicants to be 
significantly correlated with the titer of virus neutralizing antibodies, 
as determined by conventional virus neutralization assays. (See, for 
example experimental results presented in Sections 5-7, infra). This may 
be due to a decrease in the binding of non-neutralizing antibody. Hence 
assays utilizing the present perturbed antigens are useful for diagnostic 
and/or prognostic prediction of the immuno-competent status of a patient 
with respect to a particular virus. 
The method of the invention for detecting virus neutralizing antibodies in 
an aqueous sample comprises: 
(a) contacting a ligand which comprises a virus, viral antigen or fragment 
thereof having a perturbed oligosaccharide moiety with an aqueous sample 
suspected of containing virus neutralizing antibodies in an assay system 
selected from the group consisting of an enzyme-linked immunosorbent 
assay, a radioimmunoassay, an agglutination assay, and an 
immunofluorescence assay and the like; and 
(b) detecting any reaction with the ligand; in which any reaction with the 
ligand indicates the presence of neutralizing antibodies in the sample. 
When quantitating the virus neutralizing antibodies is desired the method 
further comprises: comparing any reaction of ligand and antibodies to that 
of a standard. 
5.2.1.1. DISSOCIATION AND REASSOCIATION OF IMMUNE COMPLEXES 
In certain instances, for example, during an ongoing viral infection, virus 
or virus fragments may be present in varying amounts in serum or plasma 
samples. These virus or virus fragments could be bound or associated with 
neutralizing and non-neutralizing antibodies in immune complexes. 
Consequently, the titer obtained using the present perturbed antigens in 
in vitro assays may be artifically high. Hence, according to a further 
improved embodiment of the present invention, such immune complexes are 
dissociated prior to performing diagnostic assays to separate sera having 
immune complexes from those not having immune complexes. Dissociation of 
immune complexes can be accomplished by methods known to those of skill in 
the art including but not limited to: use of chaotropic agents such as 
perchlorate (ClO.sub.4 .sup.-), thiocyanate (SCN.sup.-), etc., denaturing 
agents such as guanidine hydrochloride, urea, etc. and use of variation of 
pH, and the like. After dissociation, the liberated antibodies are 
contacted with perturbed antigens according to the present invention under 
conditions which permit association or reassociation with neutralizing 
antibodies. 
According to yet another embodiment of the invention, 
dissociation-reassociation of immune complexes present in aqueous samples 
of body fluids may be used to detect and/or quantitate the presence not 
only of neutralizing antibodies, but also of antibodies specific to 
antigens associated with the pathogenesis of autoimmune diseases which may 
be bound or associated with such antibodies in immune complexes. Thus 
according to this embodiment of the invention, antibodies present in 
immune complexes in an aqueous sample are detected by: (1) dissociating 
any immune complexes which are present in the aqueous sample; (2) 
contacting the dissociated complexes with a ligand which comprises an 
antigen associated with pathogenesis of an auto-immune disease in an assay 
selected from the group consisting of an enzyme-linked immunosorbent assay 
a radioimmunoassay, an agglutination assay and an immunofluorescence 
assay; and (3) detecting any reaction with the ligand, in which any 
reaction indicates the presence of antibody in the sample. Alternatively, 
antigens in immune complexes in aqueous samples may be detected by: (1) 
dissociating any immune complexes which are present in the aqueous sample; 
(2) contacting the dissociated complexes with a ligand which comprises an 
antibody specific for an antigen associated with pathogenesis of an 
auto-immune disease in an assay selected from the group consisting of an 
enzyme-linked immunosorbent assay a radioimmunoassay, an agglutination 
assay and an immunofluorescence assay; and (3) detecting any reaction with 
the ligand, in which any reaction indicates the presence of antigen in the 
sample. 
5.2.2. PROPHYLACTIC AND THERAPEUTIC APPLICATIONS 
According to another embodiment of the present invention, the viruses, 
viral antigens and fragments thereof having a perturbed oligosaccharide 
moiety obtained either by chemical or enzymatic oxidation methods 
described in section 5.1 are used as immunogens in vaccine formulations to 
stimulate an active, protective immune response in a vaccinated host. 
When a whole virus having an oxidized oligosaccharide moiety is used, it is 
necessary to use either an attenuated or avirulent virus or an inactivated 
virus. An inactivated virus is obtained by treatment of a virus with 
various chemicals such as formaldehyde; then an oligosaccharide is 
perturbed using any of the methods described above in Section 5.1. 
Alternatively, an inactivated virus having an oxidixed oligosaccharide 
moiety is obtained by chemical oxidation of a virus, for example, using 
periodic acid as described in Section 5.1. Such attenuated or inactivated 
viruses having an oxidized oligosaccharide moiety induce antibodies that 
are more effective at neutralizing viral infections than conventionally 
attenuated or inactivated viruses. 
Subunit vaccines containing only the necessary and relevant immunogenic 
material such as capsid glycoproteins of non-enveloped icosohedral viruses 
or the peplomers (glycoprotein spikes) of enveloped viruses or immunogenic 
fragments thereof can also be prepared in which the oligosaccharide moiety 
of the glycoprotein or fragment thereof is perturbed according to the 
present invention. Subunit vaccines can be prepared by isolating the 
relevant subunit from highly purified viral fractions or using recombinant 
DNA technology and perturbing the oligosaccharide moiety of the relevant 
immunogenic subunit as described in Section 5.1. 
The vaccine formulations which stimulate an active immune response for 
prophylaxis of viral infections can be prepared by mixing the virus, viral 
antigen or fragment thereof having a perturbed oligosaccharide moiety in a 
carrier suitable for use in vivo. In order to enhance the immunological 
response of the host, the immunogenic virus, antigen or fragment thereof 
may be formulated with a suitable adjuvant. Suitable adjuvants include, 
but are not limited to: aluminum hydroxide, surface active substances, 
lysolecithin, pluronic polyols, polyanions, peptides including but not 
limited to muramyl peptides, and oil emulsions. 
According to another alternate embodiment, a vaccine formulation to 
stimulate an active immune response for prophylaxis of viral infections is 
prepared by coupling a virus, viral antigen or fragment thereof having a 
perturbed oligosaccharide moiety to an immunogenic peptide or compound in 
order to enhance or potentiate the immunological response of the host. 
According to yet another embodiment, the vaccine formulation can be 
prepared as a multivalent vaccine. To this end, a mixture of different 
viruses, viral antigens or fragments thereof each of which contains a 
perturbed oligosaccharide moiety and which is capable of eliciting an 
immune response against a different viral pathogen can be mixed together 
in one formulation. 
Many methods can be used to introduce the vaccine formulations described 
above into a host. These include, but are not limited to: intradermal, 
intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal 
routes of administration. 
According to another embodiment of the present invention, the viruses, 
viral antigens and fragments thereof having a perturbed oligosaccharide 
moiety are used in a variety of methods to prepare neutralizing antibodies 
which can be administered to confer short-term passive immunity for 
prophylaxis and/or therapy of viral infections. In one mode of this 
embodiment, a virus, viral antigen or fragment thereof having a perturbed 
oligosaccharide moiety is used as an immunogen in any technique that 
provides for the production of antibody molecules by continuous cell lines 
in culture. For example, the present immunogens can be utilized in the 
hybridoma methods originally developed by Kohler and Milstein, and 
reviewed by them in Sci. Amer. 243: 66-74 (1980) as well as in the human 
B-cell hybridoma methods described by Kozbor et al., Immunology Today 4: 
72 (1983) and the EBV-hybridoma methods for producing human monoclonal 
antibodies described by Cole et al., in Monoclonal Antibodies and Cancer 
Therapy, Alan R. Liss, Inc., pp. 77-96 (1985) and the like. The monoclonal 
antibodies produced provide a readily available, consistent source of 
neutralizing antibodies specific for relevant virus which can be 
administered for passive immunization. 
This embodiment of the invention encompasses a method for preparing a 
composition for administration to an animal or a human to confer 
short-term passive immunity or for prophylaxis or therapy of a 
viral-induced infection comprising: harvesting monoclonal antibodies 
produced by a hybridoma cell line formed by fusing a myeloma or hybridoma 
cell and a cell capable of producing antibody against a virus, viral 
antigen or fragment thereof having a perturbed oligosaccharide moiety. It 
further encompasses a method for preparing a composition for 
administration to an animal or a human to confer short-term passive 
immunity or for prophylaxis or therapy of a viral-induced infection 
comprising: harvesting monoclonal antibodies produced by a lymphocyte cell 
line formed by transformation by an EBV virus of a mammalian lymphocyte 
cell capable of producing antibody against a virus, viral antigen or 
fragment thereof having a perturbed oligosaccharide moiety. Additionally 
it encompasses a method for preparing a composition for administration to 
an animal or a human to confer short-term passive immunity or for 
prophylaxis or therapy of a viral-induced infection comprising: (a) 
contacting a sample containing anti-viral antibodies with an antigen 
comprising a virus, viral antigen or fragment thereof having a perturbed 
oligosaccharide moiety to form an antibody-antigen complex; (b) separating 
the antibody-antigen complex from the sample; and (c) dissociating the 
antibody-antigen complex to obtain a purified antiviral antibody 
composition. 
In another mode of this embodiment, a virus, viral antigen or fragment 
thereof having a perturbed oligosaccharide moiety is used in a screening 
assay to identify those viral-specific monoclonal antibodies which 
although known in the art have not been recognized as neutralizing 
antibodies. Hence this method screens for and identifies neutralizing 
monoclonal antibodies which can then be administered for passive 
immunization. 
In yet another alternative mode of this embodiment of the present 
invention, a virus, viral antigen or fragment thereof is used to prepare 
neutralizing antibodies from serum, plasma or fractions of immunoglobulins 
derived therefrom. For example, a virus, viral antigen or fragment having 
a perturbed oligosaccharide moiety is immobilized and used in a 
preparative affinity chromatography format to isolate relevant 
neutralizing antibodies from serum or plasma by the formation of immune 
complexes. The polyclonal neutralizing antibodies are then separated from 
the immune complexes by conventional techniques. 
The neutralizing antibodies which react specifically with the compositions 
of the present invention containing a perturbed oligosaccharide moiety can 
be formulated to confer short-term passive immunity to the host. Adjuvants 
are not needed in this type of preparation because the object is not to 
stimulate an immune response, but rather to inactivate or bind a viral 
pathogen. Thus, any suitable pharmaceutical carrier can be used. Passive 
immunization using such preparations can be utilized on an emergency basis 
for immediate protection of unimmunized individuals exposed to special 
risks of viral infections. Additionally, such preparations can be used 
prophylactically for viral infections such as measles and hepatitis. 
In its most general form, the method of this embodiment of the invention 
encompasses a method for protection of an animal or a human from an 
infection induced by a virus, comprising: administering to an animal or 
human an effective amount of a vaccine formulation which comprises a 
virus, viral antigen or fragment thereof having a perturbed 
oligosaccharide moiety. 
Such formulations can be administered to a host by routes including but not 
limited to: intradermal, intramuscular, intraperitoneal, intravenous and 
subcutaneous. 
5 3. VIRUSES AND VIRAL ANTIGENS 
The viruses, viral antigens and fragments thereof which are intended to be 
encompassed by the present invention include a wide variety of viruses 
such as DNA and RNA viruses including but not limited to retroviruses. 
Specific examples include: DNA viruses such as: Adenoviridae such as 
adenoviruses subgroups B, C, D, E, F and G, etc; Herpesviridae such as 
herpes simplex I and II, cytomegalovirus, Epstein-Barr virus, 
varicella-zoster, etc.; Orthomyxoviridae such as influenza viruses, etc; 
Hepadnaviridae such as hepatitis B, hepatitis non-A, non-B, etc.; 
Parvoviridae such as parvoviruses, etc.; RNA viruses such as Togaviridae 
such as rubella, etc.; Paramyxoviridae such as measles, parainfluenza, 
respiratory syncytial virus, etc.; Flaviviridae such as dengue virus types 
1-4, yellow fever virus, tick-borne fever viruses, etc.; Rhabdoviridae 
such as rabies, vesicular stomatitis virus, Marburg-Ebola virus, etc.; 
Bunyaviridae such as Rift Valley Fever, California encephalitis virus 
group, sand fly fever virus, etc.; Arenaviridae such as Lassa fever virus, 
Junin virus, lymphocytic choriomeningitis virus, etc.; Reoviridae such as 
rotovirus, etc.; Picornaviridae such, as polio virus, coxsackieviruses, 
hepatitis A virus, rhinovirus, etc.; Retroviridae such as human 
lymphoadenopathy-associated virus (LAV, HIV, HTLV-III), human T-cell 
lymphotrophic virus types I, II, III, feline leukemia virus, etc. 
The following Examples are given for the purpose of illustration and not by 
way of limitation on the scope of the invention. 
In the ELISA assays described below in which the virus was covalently 
attached to the microtiter plate, the microtiter plates were pre-saturated 
using either 200 ul of 0.5% calf IgG in 0.14 M NaCl or 300 ul of 0.5% 
bovine serum albumin in 0.5 M Tris-citrate buffer containing 0.1% 
Tween-20, 20 pH 8.1 in order to eliminate the non-specific adsorption of 
virus or serum proteins or IgG. In the ELISA assays described below in 
which the virus was attached to the plate via adsorption, the virus was 
allowed to adsorb to the plate and then the plates were saturated using 
either calf IgG in 0.14 M NaCl or bovine serum albumin in 0.5 M 
Tris-citrate buffer. 
6. EXAMPLES: DETERMINATION OF NEUTRALIZING ANTIBODIES IN PURIFIED HUMAN IgG 
FROM SERA 
6.1. DETECTION OF NEUTRALIZING ANTI-CMV ANTIBODIES IN HUMAN IgG 
The following series of experiments demonstrate that ELISA assays in which 
the oligosaccharide moiety of the viral antigen was perturbed according to 
the present invention are useful for determining the titer of 
virus-neutralizing antibodies. In contrast, conventional ELISA assays in 
which the virus was either adsorbed or covalently attached without 
perturbation of the oligosaccharide moiety are not. 
The antigen used in the assays was whole cytomegalovirus (CMV) obtained 
from homogenates of cultured human fibroblast cells (MRC.sub.5) infected 
with CMV for six to eight days. The microtiter wells containing an 
equivalent amount (about 1 ug/well) of CMV were prepared for the various 
ELISA assays as follows: 
(A) ELISA'S Using Antigen With Perturbed Oligosaccharide. 
(1) Virus Covalently Attached Via Oxidized Oligosaccharide Moiety 
The carbohydrate moiety of CMV was oxidized using sodium periodate 
(NaIO.sub.4) as described in Section 5 and covalently coupled to a 
reactive amine on a side chain of the polyhydrazidostyrene of the well. 
Addition of a reactive amine group to polystyrene (or the microtiter well) 
was carried out by the method of Chin and Lanks [Anal. Biochem, 83: 709-19 
(1977)]. Polystyrene was first converted to nitrostyrene which was then 
reduced to aminostyrene. Polyaminostyrene was then converted to 
polyhydrazidostyrene in a two-step process: (1) polyaminostyrene was 
succinylated; and then (2) an amide bond was formed betwen hydrazine and 
the succinylated polyaminostyrene using carbodiimide. (0x Oligosaccharide 
Attached ELISA). 
(2) Virus Covalently Attached Via Amine Group, Oxidized Oligosaccharide 
Moiety 
The CMV virus was covalently attached to the polycarbostyrene via a peptide 
bond formed using carbodiimide to couple an amine residue of the virus to 
an activated carboxyl of the polycarbostyrene. The carbohydrate moiety of 
the virus was then oxidized in situ using NaIO.sub.4 as described in 
Section 4. (Amine Attached Oligosaccharide Ox ELISA). 
(B) Conventional ELISA'S Using Antigen with Non-Perturbed Oligosaccharide. 
(1) Virus Covalently Attached Via Amine Group 
CMV was attached using carbodiimide to form a peptide bond between an amine 
group of an amino acid residue of the virus and a free carboxyl group of a 
polycarbostyrene of the well. (Amine Attached ELISA). 
(2) Virus Non-Covalently Attached 
CMV was simply non-covalently fixed by adsorption onto a non-modified 
polystyrene well. (Adsorption ELISA). 
Partially purified human IgG was obtained from human serum samples using 
conventional ammonium sulfate precipitation techniques. After 
precipitation, the tubes were centrifuged at 12,000 rpm for 5 minutes. The 
supernatant was discarded, the pellet was washed, recentrifuged and 
resuspended in 0.14 M NaCl. The partially purified human IgG samples were 
serially diluted in Buffer I of the following composition: 
______________________________________ 
Calf IgG 0.5% 
NaCl 0.14 M 
Glycine 0.1 M 
Sodium borate 0.05 M 
Synperonic PE/L62 0.10% (v/v), 
______________________________________ 
adjusted to pH 8.1 with 1.0 M HCl. 100 uI of each dilution was distributed 
in the wells of the plate containing the CMV. After two hours incubation 
at 37.degree. C., the plates were washed in the Buffer I, but without calf 
IgG. 
Then 100 ul of a goat serum solution containing antihuman IgG labeled with 
alkaline phosphatase, diluted to 1/1000 in the following Buffer II, was 
introduced into each well: 
______________________________________ 
Bovine serum albumin 1.0% 
NaCl 0.14 M 
Glycine 0.1 M 
Borate 0.05 M 
Synperonic PE/L62 0.10% (v/v) 
______________________________________ 
After a contact time of one hour at 37.degree. C., the wells were washed 
with Buffer II but without bovine serum albumin (BSA). The enzymatic 
activity was determined at 37.degree. C. using p-nitrophenyl phosphate as 
substrate at a concentration of 0.2% (p/v) dissolved in a buffer 
containing: 2-amino-2-methyl-1-propanol (0.625 M) and MgCl.sub.2 (2.0 mM), 
pH 10.25. The optical density was measured at 405 nm either every five 
minutes during a period of 30 minutes or after 30 minutes of incubation. 
A conventional CMV infective power neutralization assay was performed using 
MRC.sub.5 cells in culture as described by Krech et al., Z. Immun.-Forsch, 
Bd. 141S: 411-29 (1971). 
Results are illustrated in Table 1. The antibody titer obtained using the 
ELISA assays and the conventional virus neutralization assays were 
calculated as a protein concentration of 1 mg/ml of non-diluted IgG 
sample. Table 2 presents the linear correlation coefficients obtained when 
the titers obtained by the various assays were compared. 
TABLE 1 
______________________________________ 
TITERS OF CMV-NEUTRALIZING 
ANTIBODIES IN PURIFIED IgG 
Amine 
Attached 
Ox Oligo- Oligo- 
saccharide 
saccharide 
Amine Adsorp- 
Neutral- Attached Ox Attached 
tion 
IgG ization ELISA ELISA ELISA ELISA 
Sample 
Titer Titer Titer Titer Titer 
______________________________________ 
1 128 50 &lt;50 400 67 
2 256 50 &lt;50 200 144 
3 256 100 100 400 519 
4 512 400 100 800 1087 
5 64 &lt;50 &lt;50 50 54 
6 128 50 50 200 67 
7 256 200 200 800 432 
8 465 364 181 727 2780 
9 621 485 485 485 1005 
10 512 400 400 &gt;1600 2923 
11 128 200 100 400 387 
12 256 100 100 1600 593 
13 64 &lt;50 50 54 100 
14 256 50 50 455 400 
______________________________________ 
TABLE 2 
__________________________________________________________________________ 
COMISONS OF ASSAYS FOR 
CMV-NEUTRALIZING ANTIBODIES 
Linear Correlation Coefficients 
Amine 
Attached 
Adsorption 
Ox Oligo- 
Amine Oligo 
Neutral 
ELISA saccharide 
Attached 
saccharide 
Assay ization 
Attached 
ELISA ELISA Ox ELISA 
__________________________________________________________________________ 
Neutralization 
1 
Adsorption 
0.74 1 
ELISA 
Ox Oligo- 
0.90 0.75 1 
saccharide 
Attached ELISA 
Amine Attached 
0.52 0.62 0.41 1 
ELISA 
Amine Attached 
0.78 (0.87) 
0.65 (0.64) 
0.82 (0.93) 
0.46 (0.48) 
1 
Oligosaccharide 
Ox ELISA.sup.a 
__________________________________________________________________________ 
.sup.a The number in parentheses represents the correlation coefficient 
obtained when one aberrant titer value is discarded. 
As demonstrated in Tables 1 and 2, the titer obtained using ELISA assays 
according to the present invention, i.e., the Ox Oligosaccharide Attached 
ELISA and the Amine Attached oligosaccharide Ox ELISA was highly 
positively correlated with the titer obtained using the conventional 
Neutralization Assay (correlation coefficients, respectively: 0.90 and 
0.87). On the other hand, the titer obtained using the conventional Amine 
Attached ELISA showed no significant correlation with the titer of 
neutralizing antibody (correlation coefficient: 0.52). The Amine Attached 
ELISA showed much weaker, non-significant correlation with titer of 
neutralizing antibody (correlation coefficient: 0.74). 
Table 2 demonstrates further that there was a significant positive 
correlation between the titer obtained using the Ox Oligosaccharide 
Attached ELISA and the Amine Attached Oligosaccharide Ox ELISA 
(correlation coefficient: 0.93). At the same time, however, there was no 
significant correlation observed between the titers obtained using the 
Adsorption ELISA and the Amine Attached Oligosaccharide 0x ELISA, or the 
Amine Attached Oligosaccharide Ox ELISA and the Amine Attached ELISA. This 
indicates that the significant correlation observed between the titers of 
neutralizing antibody using the Neutralization Assay and both the Amine 
Attached Oligosaccharide Ox ELISA and the Ox Oligosaccharide Attached 
ELISA is not related to the method of covalent attachment, but rather may 
be related to the perturbation of the oligosaccharide moiety achieved by 
oxidation. Moreover, these results suggest further that it does not matter 
whether the oligosaccharide perturbation occurs prior to or following 
covalent attachment of the virus to the microtiter well. 
6.2. REPRODUCIBILITY OF NEUTRALIZING ANTIBODY TITER 
The following experiment demonstrates the reproducibility of results 
obtained using an ELISA assay in which the virus was covalently attached 
to a insoluble support via an oxidized carbohydrate moiety of the virus. 
A series of ELISA assays to determine the titer of neutralizing anti-CMV 
antibodies was performed as described in Section 6.1 in which CMV was 
covalently attached to a reactive amine on a side chain of an insoluble 
support via an oxidized carbohydrate moiety of the CMV antigen. The 
samples used were purified IgG obtained from the same serum samples used 
for the experiments described in Section 6.1. One set of ELISA's were 
performed on one aliquot of purified IgG's, and a duplicate set of assays 
were performed on another aliquot of the same IgG's some 171/2 months 
later. The samples were stored frozen at -70.degree. C. during the 
interim. Results are presented in Table 3. 
TABLE 3 
______________________________________ 
REPRODUCIBILITY OF CMV 
NEUTRALIZING ANTIBODY ASSAY 
Antibody Titer - ELISA Virus Covalently 
Sample Attached Via Oxidized Oligosaccharide 
No. Experiment 1 Experiment 2 
______________________________________ 
1 50 40 
2 50 40 
3 100 160 
4 400 320 
5 &lt;50 160 
6 50 40 
7 200 160 
8 364 290 
9 485 388 
10 400 640 
11 200 160 
12 100 80 
13 &lt;50 80 
14 50 80 
______________________________________ 
As demonstrated in Table 3, the results of antibody titers obtained using 
the ELISA assay in which the virus was covalently attached via a perturbed 
oligosaccharide moiety are highly reproducible. 
7. EXAMPLE: DETECTION OF NEUTRALIZING ANTIBODIES IN SERUM SAMPLES 
The following series of assays demonstrate that an ELISA assay in which the 
oligosaccharide moiety of a virus antigen was perturbed is useful for 
determining the titer of neutralizing antibody in human serum samples. 
An ELISA assay was performed as described in Section 6.1 in which the 
oligosaccharide moiety of the CMV virus was oxidized and covalently 
coupled to a hydrazido group on the polyhydrazidostyrene of the microtiter 
well. A conventional ELISA assay was performed as described in Section 6.1 
in which the CMV was merely adsorbed to the microtiter well. A virus 
neutralization assay as described in Section 6.1 was also performed. 
Results of all three assays are compared in Table 4. 
TABLE 4 
______________________________________ 
Oxidized 
Serum Adsorption Neutralization 
Oligosaccharide 
Sample 
ELISA Assay Attached ELISA 
No. Titer.sup.a Titer Titer.sup.b 
______________________________________ 
1 6400 160 125 
2 26600 320 250 
3 102400 640 2000 
4 25600 640 1000 
5 102400 2560 4000 
6 25600 2560 4000 
______________________________________ 
.sup.a Correlation coefficient between Neutralization Assay Titer and 
Adsorption ELISA Titer: +0.37. 
.sup.b Correlation coefficient between Neutralization Assay Titer and 
Oxidized Oligosaccharide Attached ELISA Titer: +0.96. 
As demonstrated in Table 4, there was a highly significant positive 
correlation between the titer of antibodies in human serum samples 
measured by the Neutralization Assay and by an ELISA assay in which the 
oligosaccharide moiety of CMV was oxidized and covalently coupled to the 
microtiter well Thus using polyclonal sera, this ELISA assay is 
"predictive" of the immunocompetent status of the patient. In contrast, no 
correlation was observed between the antibody titer measured by the 
Neutralization Assay and that obtained using a conventional ELISA in which 
non-perturbed CMV virus was merely adsorbed to the microtiter well 
8. EXAMPLE: PERTURBATION OF OLIGOSACCHARIDE MOIETY OF VIRUS AND ATTACHMENT 
OF VIRUS TO A SOLID SUPPORT 
As suggested by results presented in Section 6.1 above, when a perturbed 
antigen is used to determine the titer of neutralizing antibodies in an 
ELISA format in which the antigen is covalently attached to the microtiter 
well, it does not matter whether the oligosaccharide moiety is perturbed 
before or after covalent attachment to the microtiter well. The following 
series of experiments was performed to investigate the effect of 
perturbation of the oligosaccharide moiety according to the present 
invention upon the ability of the virus to attach to a microtiter plate. 
The oligosaccharide moiety of CMV virus was perturbed by oxidization using 
NaIO.sub.4 for 16 hours at 4.degree. C. as described in Section 5. ELISA 
assays for anti-CMV antibodies were conducted as described in Section 6.1, 
in which: (1) CMV having a perturbed oligosaccharide moiety in PBS was 
adsorbed to a polystryene microtiter plate. (2) CMV having a perturbed 
oligosaccharide moiety was covalently coupled via the carbohydrate moiety 
to a reactive amine group of a polyhydrazidostyrene microtiter plate in 
the presence of phosphate buffer containing 0.1% Tween-20 (PBT). PBT was 
used to prevent non-covalent adsorption of CMV to the polyhydrazidostyrene 
plate. (3) Non-perturbed CMV in PBS was adsorbed to a polystyrene 
microtiter plate. 
Results are illustrated in Table 5. 
TABLE 5 
______________________________________ 
COMISON OF ELISA TITERS OF CMV 
WITH PERTURBED AND NON-PERTURBED 
OLIGOSACCHARIDE 
Ox Ox Non-Ox 
CMV CMV CMV 
IgG Adsorbed Covalently 
Adsorbed 
No. in PBS.sup.a in PBT.sup.b 
in PBS.sup.a 
______________________________________ 
Y 287 4000 4000 8000 
Y1096 1000 1000 8000 
______________________________________ 
.sup.a CMV either native or having a perturbed oligosaccharide moiety in 
PBS (phosphate buffer saline, pH 7.4) was immobilized by adsorption onto 
polystyrene microtiter plates. 
.sup.b CMV having a perturbed oligosaccharide moiety in PBT (50 mM 
phosphate buffer, 0.1% Tween20, pH 6.0) was covalently coupled to 
polyhydrazidostyrene of microtiter plates. 
As demonstrated in Table 5, there was no difference in ELISA titers 
obtained in which a perturbed antigen was either covalently attached or 
merely adsorbed to the micro-titer well. When the perturbed CMV was 
incubated in the microtiter wells in the presence of PBT, the titer 
obtained was zero because the perturbed virus does not adsorb in the 
presence of Tween-20 (results not shown). 
9. EXAMPLE: PROTECTIVE IMMUNE RESPONSE AFFORDED BY AINFLUENZA VIRUS 
HAVING PERTURBED OLIGOSACCHARIDE MOIETY 
The following example demonstrates that in vivo administration of 
parainfluenza virus (PIV) having a perturbed oligosaccharide moiety 
according to the present invention not only elicted formation of 
neutralizing antibodies, but also protected mice against a subsequent 
challenge with live virus. 
9.1. NON-INFECTIVITY OF AINFLUENZA HAVING A PERTURBED OLIGOSACCHARIDE 
MOEITY 
Parainfluenza virus (PIV) type 1 Sendai was cultured in the allantoic 
cavity of 8 day old embryonated eggs. After 3 days, the allantoic fluid 
was collected and concentrated by ultracentrifugation and ultrafiltration. 
PIV was purified by 2 successive cycles of centrifugation at 100,000 
.times. g for 4 hours on a linear sucrose gradient 20-60% (w/v). The yield 
and purity of virus were assayed by determination of hemagglutination 
activity, infectious titer, protein content and electrophoretic profile on 
polyacrylamide gel electrophoresis (PAGE). 
Preliminary experiments were performed to investigate the infectivity of 
PIV having an oxidized oligosaccharide moiety. The carbohydrate moiety of 
the whole purified PIV virion was oxidized using NaIO.sub.4 as described 
in Section 5.1. The reaction was stopped by the addition of sodium 
sulphite. The oxidized virus was dialysed extensively against PBS at 
4.degree. C. (3 changes). 
Infectivity of PIV inactivated by a conventional method using 
beta-propiolactone was also determined for comparison. Beta-propiolactone 
(Fluka) was added (final concentration 0.05% v/v) to purified PIV (2 mg 
protein/ml) in 100 mM sodium borate-HCl, pH 9.0, and the mixture incubated 
for 2 hours 37.degree. C. At that time, fresh beta-propiolactone was added 
(final concentration 0.05% v/v) and the mixture incubated for an 
additional 2 hours. The virus was then dialysed extensively against PBS at 
4.degree. C. (3 changes). 
Another sample of purified PIV was first conventionally inactivated using 
beta-propiolactone and then the oligosaccharide moiety oxidized as 
described above. The inactivated oxidized PIV was then extensively 
dialysed against PBS at 4.degree. C. (3 changes). 
Untreated PIV, incubated for 4 hours in 100 mM sodium borate-HCl, pH 9.0, 
at 37.degree. C. and then dialysed extensively against PBS, served as the 
control. 
The yield of virus obtained following each of the above treatments was 
determined by estimation of the protein content following treatment. 
Yields were 50%, 75% and 52% of control respectively for oxidized, 
inactivated and inactivated-oxidized PIV. After adjusting all preparations 
to the same protein content, 50 ul aliquots of each were tested for 
infectivity as follows: 
A 50 ul aliquot of a primary culture of Rhesus monkey kidney cells 
(3.times.10.sup.5 cells/ml) was seeded into 96 well Falcon micro-titer 
plates. Once the cells were attached, 50 ul of a series of 10-fold 
dilutions of the treated PIV samples were added to the wells (4 
wells/dilution). The infectious titer of PIV represents the last dilution 
which still resulted in (a) characteristic cytopathic effects of PIV on 
the monkey kidney cells; and (b) typical hemadsorptic pattern using guinea 
pig red blood cells using an assay described in Vogel et al., Science 
126:358-59 (1957) adapted for use in microtiter plate. 
Results obtained are presented in Table 6. 
TABLE 6 
______________________________________ 
INFECTIVITY OF AINFLUENZA VIRUS (PIV) 
Infectious PIV Titer.sup.a 
Treatment 
Conventional 
Sample Oligosaccharide 
Conventional 
Inactivation 
No. Control Oxidation Inactivation 
& Oxidation 
______________________________________ 
l &gt;10.sup.9 
&lt;10 &lt;10 &lt;10 
2 10.sup.5 
&lt;10 &lt;10 &lt;10 
______________________________________ 
.sup.a Infectious titer of dilutions of 50 ul of treated samples of PIV 
were obtained after adjusting all samples to the same protein content as 
described in the text. The titer represents the last dilution which still 
resulted in (a) characteristic cytopathic effects of PIV in cultured 
monkey cells; and (b) typical hemadsorption pattern using guinea pig red 
blood cells. See text for details. 
As demonstrated in Table 6, oxidation of the oligosaccharide moiety of PIV 
effectively destroyed the infectivity of the virus. In fact, oxidation of 
the carbohydrate moiety of the PIV virion was as effective as conventional 
inactivation using beta-propiolactone. 
9.2. INDUCEMENT OF NEUTRALIZING ANTIBODIES 
The following experiment demonstrates that intraperitoneal administration 
of PIV having a perturbed oligosaccharide moiety effectively induced the 
formation of neutralizing antibodies in experimental animals. 
Sixty-six 4 week old female CBA/JICO mice were divided into one group of 
six animals (control group) and 3 groups of 20 experimental animals 
(Series A, B, and C). Animals in each Series were further subdivided into 
four treatment groups: Group 1 (control) untreated PIV; Group 2, PIV 
having an oxidized oligosaccharide moiety; Group 3, PIV conventionally 
inactivated using beta-propiolactone; and Group 4, PIV conventionally 
inactivated using beta-propiolactone and having an oxidized 
oligosaccharide moiety. Animals in Series A, Series B and Series C 
received respectively-one (on day 0) two (on days 0 and 14) and 3 (on days 
0, 14 and 28) intraperitoneal injections of the appropriate PIV 
preparations (70 ug PIV in 100 ul PBS and 100 ul Fruend's complete 
adjuvant). 
Serum samples were obtained from animals via retro-orbital puncture 14 days 
after the last injection of PIV preparations for each Series. Antibody 
titer of the serum samples was evaluated using three different assays as 
follows: 
(1) Oxidized Oligosaccharide Attached ELISA. An ELISA assay was performed 
as described in Section 6.1. in which the antigen used comprised PIV 
having an oxidized oligosaccharide moiety covalently coupled to a 
hydrazido group on the polyhydrazido styrene of the microtiter well. 
(2) Conventional Adsorption ELISA. A conventional ELISA assay was performed 
in which the PIV was fixed merely by adsorption onto the non-modified 
polystyrene microtiter well. Briefly, 0.1 ug purified PIV in PBS, pH 7.2, 
was allowed to adsorb onto the wells by incubation for 16 hours at room 
temperature. The wells were then washed with PBS containing 0.05% Tween 20 
(PBT-0.05). 
Serum samples, diluted in PBT-0.05 100 ul were distributed in the wells of 
the plates containing PIV. The sence and titer of anti-PIV antibody was 
determined as described in Section 6.1. using goat anti-mouse IgG labeled 
with alkaline phosphatase. 
(3) Conventional PIV Neutralization Assay. A virus neutralization assay was 
performed as follows: Serial 2-fold dilutions of serum were incubated for 
1 hour at 37.degree. C. in the presence of an equal volume of virus 
containing 30-100 times the dose of virus capable of producing a 
cytopathic effect on 50% of cells in tissue culture. (TCID.sub.50) 100 ul 
of the mixture were then added to each well of Falcon microtiter plates 
containing 50 ul of a suspension of Rhesus monkey kidney cells at 
3.times.10.sup.5 cells/ml. Incubation was carried out at 30.degree. C. in 
a humid atmosphere of air/CO.sub.2 (95/5). The neutralizing titer of the 
serum represents the highest dilution which still inhibits the cytopathic 
effect of the virus. 
Results obtained using all three assays are illustrated in FIG. 1 (A-D). As 
indicated in FIG. 1, antibody titer rose appreciately from day 18-32 in 
all animals which received PIV preparations as immunogen. In mice which 
received either untreated PIV or PIV having an oxidized oligosaccharide 
moiety as immunogen, the rise in neutralizing antibody titer at days and 
28 followed the same pattern when assayed by the oxidized oligosaccharide 
attached assay and conventional neutralization assays (FIG. 1A, B). In 
mice which received conventionally inactivated virus, there was a lag in 
the neutralizing antibody titer (FIG. 1C). In mice which received PIV 
which was both conventionally inactivated and in which the oligosaccharide 
moiety was oxidized as immunogen, the titer of neutralizing antibody 
obtained using virus neutralization assay remained low. 
From results presented in FIG. 1, it is clear that PIV having an oxidized 
oligosaccharide moeity is effective in eliciting a significant 
neutralizing antibody response when administered in vivo. This response 
occurred earlier than that elicited by PIV inactivated by conventional 
methods using beta-propiolactone. 
In order to determine persistence of neutralizing antibody elicited by PIV 
having an oxidized oligosaccharide moiety, a series of experiments was 
performed in which animals received two-three intraperitoneal injections 
of the perturbed PIV as immunogen at day 0, 14 and 28. Serum samples were 
collected and the neutralizing antibody titers was assayed using 
biological neutralization test. Results obtained are illustrated in FIG. 
2. 
As demonstrated in FIG. 2, three intraperitoneal injections of oxidized PIV 
were necessary to maintain maximum neutralizing antibody titer for up to 
42 days. 
9.3. PROTECTIVE IMMUNE RESPONSE 
The critical requirement for a successful vaccine formulation is the 
ability not only to induce neutralizing antibody, but more importantly, to 
elicit a protective immune response against infectious virus. The 
following experiment demonstrates that animals previously immunized using 
PIV having an oxidized oligosaccharide moeity were effectively protected 
against subsequent challenge with infectious PIV virus. 
The same group of 66 female mice as described in Section 9.2 were used as 
experimental animals. An additional group of 12 mice which were not 
immunized served as controls. On the 14th day following the last 
intraperitoneal administration of the appropriate PIV preparation, all 
animals from Series A, B and C, were challenged intranasally with 10.sup.3 
TCID.sub.50 of untreated PIV. Control mice were similarly challenged. Four 
days following challenge with untreated (live) PIV, animals were 
sacrificed and the presence of infectious virus was evaluated in lung 
tissues as follows: 
Lung tissues were homogenized in minimal essential medium (MEM) containing 
trypsin at a concentration of 5 ug/ml (MEM-trypsin). Aliquots of the lung 
homogenates were diluted in MEM-trypsin and added to culture tubes 
containing 1.times.10.sup.4 MA 10.sub.4 cells (a cell line derived from 
Rhseus monkey kidney cells). After the cells were cultured for three days, 
the virus titer was determined by hemadsorption. Comparison of the titer 
obtained in pulmonary homogenates of non-immunized control animals 
permitted the degree of protection to be estimated. 
In non-immunized mice, virus multiplication in the lungs resulted in a PIV 
titer of 1.5.times.10.sup.6 TCID.sub.50 in lung homogenates four days 
post-challenge. In contrast, in animals previously immunized with one 
intraperitoneal injection of PIV having an oxidized carbohydrate moiety, 
the titer of virus recovered was only 7.5.times.10.sup.2 TCID.sub.50. In 
animals immunized with either 2 or 3 intraperitoneal injections, no virus 
was detected in lungs, four days following challenge with infectious 
virus. 
In animals which had been previously immunized with one, two or three 
intraperitoneal injections of either conventionally inactivated PIV or 
conventionally inactivated PIV having an oxidized carbohydrate moiety, no 
virus could be detected in lung homogenates at any time post-challenge 
with infectious virus. Thus it appears that PIV having an oxidized 
oligosaccharide moiety either with or without conventional inactivation is 
very effective in elicting a protective immune response against infectious 
PIV. 
10. EXAMPLE: BIOLOGICAL ACTIVITY OF VIRUSES HAVING A PERTURBED 
OLIGOSACCHARIDE MOIETY 
The following experiments demonstrate that oxidation of the carbohydrate 
moiety of influenza virus (an RNA virus) and of adenovirus (a DNA virus) 
is effective in destroying infectivity and/or hemagglutination ability of 
the virus. 
The oligosaccharde moiety of influenza virus A Hong Kong 168 and of 
Adenovirus type 5 (AdV5) whole virions was oxidized according to the 
present invention as described in Section 5.1. The biological activity of 
the virions having oxidized carbohydrate moeities was compared to 
equivalent preparations of untreated virions as follows: 
(1) Hemagglutination Activity: Influenza A Hong Kong 168 (IV-AHK) having an 
oxidized carbohydrate moiety and untreated IV-AHK were cultured in 
embryonated eggs for 48 hours at 33.degree. C. Hemagglutination activity 
of virus in the allantoic fluids was determined using chick red blood 
cells. 
(2) Infectivity: Infectivity of IV-AHK was assayed using embryonated chick 
eggs. 
Infectivity of AdV5 was assayed using HeLa cells in culture. [See Vogel et 
al., Science 126:358-59 (1957); Palmer et al., Advanced Laboratory 
Techniques For Immunological Diagnosis, Public Health Service 
Immunological Series No. 6 pgs. 25-62 (1975)]. Results obtained are 
presented in Table 7. 
TABLE 7 
______________________________________ 
BIOLOGICAL ACTIVITY OF VIRUSES 
HAVING AN OXIDIZED CARBOHYDRATE MOIETY 
Oxidized 
Biological Carbohydrate 
VIRUS Activity Untreated Moiety 
______________________________________ 
A Hong Kong 
Hemagglutination 
5 .times. 10.sup.4 
200 
1 A 68 activity 
A Hong Kong 
Infectious 10.sup.12 10 
1 A 68 titer 
AdV.sub.5 Infectious 10.sup.6 10 
titer 
______________________________________ 
As demonstrated in Table 7, hemagglutination activity and infectivity of 
influenza A Hong Kong 1 A68 was effectively destroyed by oxidation of the 
carbohydrate moiety. Infectivity of Adenovirus type 5 was also effectively 
destroyed by such treatment. 
11. EXAMPLE: QUANTITATIVE DETERMINATION OF THE TITER OF NEUTRALIZING 
ANTIBODIES BY DISSOCIATION AND REASSOCIATION OF IMMUNE COMPLEXES 
As described above in section 5.2.1.1., in certain instances, such as 
during an ongoing viral infection, virus or virus fragments could be bound 
or associated with neutralizing and non-neutralizing antibodies in immune 
complexes in serum. 
In any event, determination of antibody titers before and after 
dissociation of any antigen-antibody complexes could reveal the presence 
of such immune complexes in serum samples. 
11.1. IMMUNE COMPLEXES DURING ACTIVE CMV INFECTION 
As a preliminary experiment, acid dissociation and alkaline reassociation 
of IgG samples positive and negative for anti-CMV antibodies was performed 
in order to verify that there were no adverse effects on the titer of free 
antibodies merely as a result of such dissociation-reassociatin. In order 
to dissociate complexes, aliquots of diluted serum at pH 7.2 were further 
diluted 1/50 in buffer containing 0.3 M NaCl, 1% BSA, 10 mM glycine-HCl, 
pH 2.2, and incubated at 4.degree. C. for 15 minutes. To reassociate any 
complexes, the pH of the reaction mixture was readjusted to pH 8.1 by 
addition of neutralizing buffer containing: 0.05 M Borate; 0.010 M 
glycine; 0.15 M NaCl; 10% Calf serum; 0.1% synperonic PE/L62 adjusted to 
pH 8.1 with 0.1 M HCl. An ELISA using CMV having an oxidized carbohydrate 
covalently attached to a reactive amine on a side chain of the 
polyhydrazidostyrene of the well as described in Section 6.1 above was 
performed. Results are presented in Table 8. 
TABLE 8 
______________________________________ 
Antibody Titer-Oxidized Oligosaccharide 
Non Dissociated 
Dissociated 
Sample Reassociated 
Sample pH 8.1 pH 2.2-pH 8.1. 
______________________________________ 
IgG .gtoreq.1600 .gtoreq.1600 
positive for 
anti-CMV 
Control IgG &lt;100 &lt;100 
Negative for 
anti-CMV 
______________________________________ 
As shown in Table 8, dissociation-reassociation had no detectable effect on 
free anti-CMV antibodies. 
Serum samples were obtained from a renal transplant patient before 
transplantation, during CMV infection, and following recovery. The titer 
of anti-CMV antibodies in a series of serum samples from this renal 
patient were determined using an oxidized oligosaccharide covalently 
attached ELISA as described in Section 6.1 with or without 
dissociation-reassociation. The antibody titer was also assayed using a 
conventional adsorption ELISA as described in Section 6.1. Results are 
presented in Table 9. 
TABLE 9 
______________________________________ 
Antibody Titer 
Oxidized Oligosaccharide 
Adsorption 
Covalent ELISA ELISA 
Non- Disassociated 
Non- 
Patient Disassociated 
Reassociated 
Dissociated 
Status pH 8.1 pH 2.2-pH 8.1 
pH 8.1 
______________________________________ 
No CMV in 100 100 100 
Urine 
CMV in Urine 
&lt;100 .gtoreq.1600 
.gtoreq.l2800 
No CMV in &gt;1600 .gtoreq.1600 
.gtoreq.12800 
Urine 
______________________________________ 
As demonstrated in Table 9, when CMV was detectable in urine, there was a 
significant difference in antibody titer measured with or without 
dissociation-reassociation. In contrast, when no CMV was detectable in 
urine, there was no significant different in antibody titer with or 
without disocciation-reassociation. These results strongly suggest the 
presence of immune complexes in the serum sample when CMV was detected in 
urine. 
11.2. IMMUNE COMPLEXES IN SERUM SAMPLES 
Serum samples from unknown blood donors either seropositive or seronegative 
for HIV were obtained as a gift from Drs. J. C. Chermann and F. 
Barre-Sanussi of the Institut Pasteur, Paris. The samples had been 
incubated at 56.degree. C. for 30 minutes to inactivate any HIV virus 
present. 
Antibody titers of aliquots of the sera were assayed as described above 
using three different assays: (1) Oxidized Oligosaccharide Attached ELISA 
in which there was no dissociation of the serum sample; (2) oxidized 
oligosaccharide attached ELISA in which the serum sample was dissociated 
and then reassociated; and (3) a conventional virus neutralization assay 
was performed by Drs. Chermann and Barre-Sanussi at the Institute Pasteur. 
Results obtained are illustrated in Table 10. 
TABLE 10 
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SERA FROM HIV SEROPOSITIVE AND 
SERONEGATIVE INDIVIDUALS 
Oxidized Oligosaccharide 
Attached ELISA Titer 
Disassociated 
Serum Non-Disassociated 
Reassociated 
Neutralization 
No. pH 8.1 pH 2.2-pH 8.1 
titer 
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1 100 100 100 
3 200 100 &lt;50 
6 &lt;100 100 &lt;50 
11 &lt;100 100 50 
12 800 400 8000 
14 &lt;100 100 200 
15 200 100 &lt;50 
16 &lt;100 100 200 
17 100 100 200 
19 &lt;100 100 50 
20 &lt;100 100 &lt;50 
13 800 400 200 
2 400 .gtoreq.1600 
200 
4 800 .gtoreq.1600 
200 
5 100 400 &lt;50 
7 800 .gtoreq.1600 
1000 
8 200 .gtoreq.1600 
200 
9 100 400 50 
10 200 .gtoreq.1600 
200 
18 100 400 100 
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As indicated in Table 10, for some sera, anti-HIV titers obtained with or 
without dissociation-reassociation were markedly different. These sera 
were considered to be immune complex positive sera. In other sera, there 
was no marked difference in anti-HIV titer obtained with or without 
dissociation-reassociation. These sera were considered immune complex 
negative. 
When all groups of sera including both immune complex positive and negative 
were grouped together, the titer obtained using the oxidized 
oligosaccharide ELISA with or without dissociation showed not correlation 
with the classical neutralization assay titer (correlation coefficient 
0.59). When only those sera which were immune complex negative were 
considered, however, there was a significant positive correlation of 
antibody titer obtained using the oxidized oligosaccharide ELISA (with or 
without dissociation-reassociation) and using conventional virus 
neutralization assay (correlation coefficients 0.99; 0.98 respectively). 
When only those sera which contained immune complexes were considered, 
there was a better correlation of titer obtained with the virus 
neutralization assay and antibody titer using oxidized oligosaccharide 
ELISA without dissociation-reassociation (correlation coefficient 0.73) 
than with oxidized oligosaccharide ELISA with dissociation (correlation 
coefficient 0.48). In either case, however, neither correlation was 
significant when sera contained immune complexes. 
The invention described and claimed herein is not to be limited in scope by 
the specific embodiments herein disclosed, since these embodiments are 
intended as illustrations of several aspects of the invention. Any 
equivalent embodiments are intended to be within the scope of this 
invention. Indeed, various modifications of the invention in addition to 
those shown and described herein will become apparent to those skilled in 
the art from the foregoing description. Such modifications are also 
intended to fall within the scope of the appended claims.