Immunotherapy for recurrent HSV infections

Immunotherapy for the treatment of Herpes Simplex Virus eye infections is disclosed. The invention involves a local therapeutic or prophylactic vaccine for the eye, comprising one or more recombinant HSV-1 glycoproteins or proteins, specifically gB and gD, in combination with at least one adjuvant to reduce the incidence of primary HSV-1 infection and/or decrease spontaneous HSV-1 ocular shedding which in turn, controls recurrent corneal disease.

I. FIELD OF THE INVENTION 
The present invention relates to the field of infectious diseases and 
ophthalmology. 
II. BACKGROUND OF THE INVENTION 
A. Incidence and Course of HSV Infection 
Herpes Simplex Virus (HSV), also known as herpesvirus hominis, is 
classified into two (2) types, 1 and 2. HSV-1 is transmitted by physical 
contact, such as kissing, and is thus spread among family members and 
friends. About half of all babies in the United States are born with IgG 
antibodies to this agent which is transmitted across the placenta. As this 
immunity dissipates, new infections are acquired until, by age 45, close 
to 70% of people have become serologically positive--most without ever 
experiencing signs of disease, others after one or several episodes of 
fever blisters or cold sores. 
In contrast, HSV-2, also called genital herpes, is transmitted during birth 
or by sexual contact. The latter's incidence rises with the number of 
sexual partners and has therefore greatly increased in today's society. 
Compared with HSV-1, genital herpes is less prevalent overall but is 
likewise cumulative with age. In addition, genital herpes has engendered 
considerable anxiety because there is tenuous evidence that it may 
contribute to the causation of cervical cancer, and because of the risk of 
vertical transmission during childbirth inducing serious disease. 
Infections with both causative agents are difficult to prevent; and there 
is as yet no proven vaccine for the prophylaxis of genital herpes. 
Moreover, an ocular vaccine for the prophylaxis of ocular HSV has not been 
tried in humans. 
Turning specifically to HSV-1, it is the most common infectious cause of 
blindness in industrial nations (1). The often prolonged ocular disease 
results in considerable visual morbidity, medical expense and loss of 
productivity in otherwise healthy individuals. Approximately 500,000 cases 
of ocular HSV-1 are diagnosed annually in the United States alone; and 25% 
to 45% of these cases may be expected to recur within 1 to 2 years after 
the primary disease episode (1). Of note, the majority of cases diagnosed 
as primary HSV are actually recurrent infections, as the patient may not 
recall the antecedent attack. Recurrence is therefore the hallmark of HSV 
infection. 
After primary HSV infection occurs, the virus can travel in the nerves to 
the neurons in the trigeminal ganglia, where it then persists throughout 
life. This critical factor presently makes the herpes simplex infection an 
incurable disease, since the virus eventually may travel back down these 
nerves and reinfect the part of the body innervated by that nerve. Various 
trigger mechanisms such as trauma, fever, sunlight exposure or stress may 
initiate the reactivation process. This latency-reactivation-recurrence 
cycle results in ocular virus shedding despite a good local ocular IgA 
response to the virus (2). Once HSV has recurred in the eye, corneal 
disease and stromal scarring can follow, resulting in corneal blindness. 
Over 1,000 corneal transplants per year are currently performed in the 
U.S. as a direct result of HSV scarring. Hence, on recovering from the 
initial HSV infection, the stage is set for reinfection from one's own 
herpes virus for the remainder of the individual's life. 
Since recurrences continue throughout the lifetime of the infected 
individual, it is clear that natural HSV infection affords insufficient 
protection against HSV recurrences. Moreover, individuals infected with 
one HSV serotype are only partially protected against subsequent infection 
with the other serotype; while individuals with non-ocular HSV-1 are not 
protected against subsequent ocular HSV-1 infection. Virus from a 
recurrent lesion on the body can be transferred to the eye, which is 
thought by some to be a common mode of contracting ocular infections. 
Because repeated recurrences of HSV do not elicit an immune response that 
prevents additional recurrences, there is a critical need to elicit a 
stronger, or perhaps a different immune response than that elicited by 
natural HSV-1 infection. 
With further regard to immune protection, it appears that both antibody and 
cell-mediated immunity (CMI) are important in the control of HSV infection 
(3, 4, 5), although CMI may play a larger role. Patients with defects in 
CMI generally have more severe infection than those with impaired humoral 
immunity (6-10); whereas patients with frequently recurring HSV have high 
titers of anti-HSV antibodies. Ophthalmologists have also demonstrated 
that patients with exuberant immune responses, such as atopes, develop the 
worst clinical manifestations of stromal herpetic keratitis. Whereas 
immunosuppressed patients, in contrast, show exacerbated epithelial 
keratitis but minimal stromal disease. Hence, immunotherapy capable of 
inducing a specific higher than normal cellular immune response is needed 
to combat recurrent ocular HSV infections. 
Another factor attributing to recurrent ocular HSV infection is the absence 
of blood vessels in the cornea. Because the cornea is devoid of blood 
vessels, systemic immune responses can be inefficient at providing 
protection from antigenic insults there (11). Consequently, local immunity 
may be particularly important in protection against ocular HSV. There is 
therefore the need for local ocular immunotherapy to augment the immune 
response and control recurrent ocular HSV infection. 
Currently, commercial HSV vaccine development is directed exclusively to 
the problem of genital HSV-2. There is minimal effort directed to combat 
ocular HSV-1. Yet the development of a therapeutic vaccine, that is, a 
vaccine to reduce HSV ocular recurrences, would greatly alleviate what is 
now the most frequent serious viral eye infection in the U.S. and a major 
cause of viral induced blindness in the world. The present invention 
satisfies this need and provides related advantages as well. The 
disclosures of all publications cited herein are expressly incorporated by 
reference. 
B. DNA Technology 
Recombinant DNA and associated technologies can be applied to effectively 
provide the large quantities of high quality bioactive HSV glycoproteins 
and proteins required for a therapeutic or prophylactic HSV vaccine. 
DNA technology involves in part, producing a replicable expression vehicle 
by the DNA recombination of an origin of replication, one or more 
phenotypic selection characteristics, an expression promoter, a 
heterologous gene insert and remainder vector. The resulting expression 
vehicle is introduced into cells by transformation and large quantities of 
the recombinant vehicle obtained by growing the transformant. Where the 
gene is properly inserted with reference to portions which govern the 
transcription and translation of the encoded DNA message, the expression 
vehicle may produce the polypeptide sequence for which the inserted gene 
codes. This process of producing the polypeptide is called "expression." 
The resulting product may be obtained by lysing the host cell, and 
recovering the product by appropriate purification. 
A wide range of host cells can be used, including prokaryotic and 
eukaryotic organisms. In addition to microorganisms, cultures of cells 
derived from multicellular organisms, whether vertebrate or invertebrate, 
may also be used as hosts. 
C. Definitions 
As used in this disclosure, the following terms are to be understood in 
relation to the following definitions. 
ADJUVANT: a substance that enhances, nonspecifically, the immune response 
to an antigen. An adjuvant is usually administered with antigen, but may 
also be given before or after antigen. Adjuvants disclosed within the 
subject invention include but are not limited to, alum, Freund's, MTP-PE, 
ISCOMs, Quil A and liposomes. 
ALUM: antigen absorbed into floccules of aluminum salts. Alum is the only 
adjuvant currently approved by the FDA for human use. 
EXPRESSION VECTOR: a vehicle used to carry inserted foreign (heterologous) 
DNA for the purpose of producing more material or a glycoprotein or 
protein product. "Expression vector" includes vectors which are capable of 
expressing the DNA sequences it contains, where such sequences are 
operably linked to other sequences capable of effecting their expression. 
Any DNA sequence which is capable of effecting expression of a specified 
DNA code disposed within the sequence is included in this term as it is 
applied to the specified sequence. In general, expression vectors of 
utility in recombinant DNA techniques are often in the form of plasmids; 
however, this invention is intended to include other forms of expression 
vectors which serve equivalent functions and which subsequently become 
known in the art. 
FREUND'S: A water-in-oil emulsion. There are two forms of Freund's 
adjuvant, depending on the presence or absence of killed Mycobacteria. 
Complete Freund's adjuvant contains Mycobacterium tuberculosis, or other 
Mycobacteria strains. Weak antigens may be rendered more immunogenic when 
incorporated in complete Freund's adjuvant. Incomplete Freund's adjuvant 
lacks Mycobacteria and is less stimulatory. 
GLYCOPROTEIN: a class of compounds in which protein is combined with 
carbohydrate. 
MTP-PE: a new proprietary adjuvant developed by CIBA and refined by Chiron 
comprising 
M-acetylmuramyl-L-alanyl-B-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn 
-glycerol-3-hydroxyphosphoryloxy)-ethylamine. It is a potent and well 
tolerated derivative of Freund's, and has proven to be much more effective 
than alum. 
ISCOH (Quil A): immunostimulating complexes comprising purified proteins 
and the glycoside Quil A to form a honeycomb like structure that exhibits 
strong adjuvant activity. 
IMMUNOTHERAPY: enhancement of an immune response by any one or more of a 
variety of adjuvants incorporating one or more antigens. 
LIPOSOMES: synthetic lipid vesicles consisting of phospholipid bilayers 
surrounding one or more aqueous compartments. Antigens can be imbedded in 
the liposomes for induction of immune responses. 
PLASMID: circular double standed DNA which, in vector form, is not bound to 
the chromosome. 
PROMOTER: a region of DNA involved in the binding of RNA polymerase to 
initiate transcription. 
VACCINE: a composition which produces active immunity. A vaccine is 
comprised of materials from microorganisms that contain antigens in an 
innocuous form with or without one or more adjuvants. The materials may 
comprise antigenic determinants and/or subunits from the microorganism and 
may be in the form of glycoproteins or proteins. 
PROPHYLACTIC VACCINE: an active immunity inducing composition given to 
naive individuals to prevent or ameliorate primary infection and prevent 
the establishment of latent infection. 
THERAPEUTIC VACCINE: an active immunity inducing composition given to 
individuals with latent or recurrent infection to reduce or minimize 
recurrences. 
SYSTEMIC VACCINE: a composition for inducing active immunity relating to 
the entire individual as distinguished from any one individual area. 
LOCAL VACCINE: a composition for inducing active immunity relating to one 
individual area as distinguished from the entire individual. 
III. SUMMARY OF THE INVENTION 
The purpose of this invention is to produce, by recombinant DNA techniques, 
an HSV glycoprotein or non-glycoprotein polypeptide, including but not 
limited to a gD-related, gB-related or Vmw65-related protein, which may be 
used as an immunogen in a vaccine to protect against HSV-1 and/or HSV-2 
infections. Vaccines made from genetically engineered immunogens should be 
safer than conventional vaccines made from attenuated virus because there 
is no risk of infection to the recipient; and specifically with the herpes 
virus, there should be no risk of cervical cancer. Alternatively, the 
genetically engineered glycoprotein or protein product may be used to 
produce antibodies for use in passive immunotherapy. 
Methods and compositions are therefore provided for the cloning and 
expression of an HSV glycoprotein or non-glycoprotein gene in single-cell 
host organisms. However, the present invention could be practiced in any 
cell line that is capable of the replication and expression of a 
compatible vector, including but not limited to, CHO and Vero cell lines. 
The invention is also intended to include expression vectors which serve 
equivalent functions as that described herein, and which become known in 
the art subsequently to this application. Also described are methods for 
culturing these novel single-cell organisms to produce the HSV gene 
product and methods for the purification of the gene product. 
A human host is then preferably inoculated with a vaccine comprising an 
immunity inducing dose of one or more HSV glycoproteins or proteins of the 
invention by the systemic route, the enteric route or by ocular route. 
When administered by the ocular route, the vaccine can be given alone or 
in combination with systemic and/or enteric vaccination. The vaccine may 
also comprise one or more adjuvants administered with, before or after the 
glycoprotein component of the vaccine. Typically, one or several 
inoculations of between about 10-1000 .mu.g each are sufficient to effect 
immunization of a human host. 
The vaccine of the invention may be conveniently utilized in liquid form, 
freeze-dried, spray dried or lyophilized form, in combination with one or 
more suitable preservatives and protective agents to protect the 
glycoproteins or proteins during processing. 
A. Antigens 
The HSV-1 glycoproteins comprising the subject immunotherapy include but 
are not limited to glycoproteins gB, gC, gD, gE, gG, gH, gK, gL, gI, 
Vmw65, ICP0 and ICP4. The HSV-1 glycoproteins may be native, purified 
native, recombinant and/or synthetic. The recombinant glycoproteins may be 
obtained by the procedure set forth below, or any equivalent procedure. 
The vaccine may comprise one or more of these glycoproteins in a dose of 
10-1000 .mu.g per inoculation. 
B. Adjuvants 
Vaccines are often administered in an emulsion with various adjuvants. The 
adjuvants aid in attaining a more durable and higher level of immunity 
using smaller amounts of antigen in fewer doses than if the immunogen were 
administered alone. The adjuvants for use in the present invention include 
but are not limited to alum, Freund's, MTP-PE and ISCOMs (Quil A). In 
addition, the vaccine may comprise a liposome or other membrane bound 
vesicle comprising one or more HSV-1 glycoproteins administered with or 
without one or more adjuvants to induce the cell mediated immune response. 
C. Immunization Routes 
The ocular route is the preferred route of inoculation; however, this 
designation as the preferred inoculation route is not meant to preclude 
any other route of administration. The vaccine can be administered by the 
ocular route either alone or in combination with systemic (intramuscular 
or subcutaneous) and enteric vaccination. The ocular route includes but is 
not limited to subconjunctival injection, surface drops, a slow-release 
device such as a collagen shield, a hydrogel contact lens or an ALZA 
"Ocusert." 
Subconjunctival vaccination is done using proparacaine for anesthesia prior 
to the injection of 0.2-0.5 ml of vaccine, in a dose of 10-1000 
.mu.g/inoculation, given in an insulin syringe and a small gauge needle. 
The injection is given in the lower cul-de-sac ensuring that the vaccine 
material remains subconjunctival and does not leak out. 
The surface drops vaccination involves placing 50 .mu.l of expressed 
glycoproteins with or without adjuvant in the conjunctival cul-de-sac and 
then rubbing the eye gently for 30 seconds while held closed. Since the 
expressed glycoprotein may be quickly cleared, the procedure should be 
repeated four times a day for five days to prolong the exposure, all of 
which comprise a single vaccination. For better retention, the tear 
drainage ducts may be temporarily blocked using collagen or other devices. 
A collagen shield may also be soaked in a concentrated solution containing 
glycoproteins and adjuvant and then placed in the eye like a contact lens. 
The lid is then closed for several days by external application of medical 
grade cyanoacrylate adhesive, during which time the antigen is 
continuously released. Alternatively, the glycoproteins may be 
encapsulated in a microcapsule and then implanted into the eye to 
facilitate continuous antigen release. 
C. Expression System For The Glycoproteins 
Consistent high expression of the HSV-1 glycoproteins from the same source 
is an important factor in the development of a human HSV vaccine. Until 
recently, studies have been hampered by the diverse expression vectors and 
the diverse viral sources of the various HSV-1 glycoproteins expressed, 
making meaningful comparison among the expressed glycoproteins difficult. 
To overcome this problem, we have individually expressed high levels of 
seven HSV-1 glycoproteins from one virus strain in a single vector system 
as outlined in detail in Section V below. 
The examples set forth below describe use of baculovirus, the polyhedron 
promoter system and insect cells as host cells. However, it would be well 
within the skill of the art to use analogous techniques to construct 
expression vectors for expression of desired glycoprotein and protein 
products in alternative host cell cultures. 
D. Test Model 
An important tool in the development of a human ocular HSV vaccine is the 
test animal model used. Our test system is the rabbit eye because HSV 
infections there very closely simulate what happens in the human eye. For 
instance, the severity of the eye disease is similar, latency is similarly 
established, and spontaneous reactivations occur much as they do in 
humans. 
The invention relates generally to immunotherapy for the treatment of HSV 
infection in a human host. The invention also relates to the expression of 
high levels of high quality bioactive HSV-1 glycoproteins or proteins from 
one virus strain in a single vector system. 
One aspect of the invention involves a systemic vaccine comprising one or 
more HSV-1 glycoproteins or proteins to decrease spontaneous HSV-1 
shedding and to reduce the incidence of primary HSV-1 infection. Another 
aspect of the invention involves a local ocular therapeutic vaccine 
comprising one or more HSV-1 glycoproteins or proteins to decrease 
spontaneous HSV-1 ocular shedding and thereby control recurrent corneal 
disease. Another aspect of the invention involves a local ocular 
prophylactic vaccine comprising one or more HSV-1 glycoproteins or 
proteins to reduce the incidence of primary HSV-1 infection. 
It is therefore a general object of the present invention to develop 
effective immunotherapy for the treatment of HSV in a host. 
It is an object of the present invention to develop effective systemic 
immunotherapy for the treatment or prevention of HSV in a human host. 
It is an object of the present invention to develop effective local 
immunotherapy for the treatment of ocular HSV in a human host. 
It is also an object of the present invention to develop a local 
therapeutic ocular vaccine to decrease spontaneous HSV ocular shedding and 
to control recurrent disease in a human host. 
It is a further object of the present invention to develop a local 
prophylactic ocular vaccine to reduce the incidence of primary HSV 
infection in a human host. 
It is still further an object of the invention to utilize an animal test 
model that closely simulates HSV infection in the human eye. 
It is another object of the present invention to individually express high 
levels of the seven HSV-1 glycoproteins from one virus strain in a single 
vector system. 
These and other objects will become readily apparent to those skilled in 
the art from the following description and appended claims. 
IV. BRIEF DESCRIPTION OF THE DRAWINGS 
The present invention will be described in connection with the accompanying 
drawings in which: 
FIG. 1 is a schematic diagram of the construction of plasmid pAC-gD1 used 
in constructing exemplary recombinant virus strains of the invention. 
FIG. 2 is a Western Blot analysis of eleven recombinant baculoviruses 
expressing HSV-1 glycoprotein in insect cells. Lane M represents molecular 
weight markers. 
FIG. 3 is a Southern Blot analysis of recombinant baculovirus DNA; a. 
Ethidium bromide staining gel; b. Autoradiogram; M. markers (IKb); P. 
plasmid-BamHI cut pAc-gD1 transfer vector; B. baculovirus recombinant 
BamHI cut DNA. Arrows indicate the location of the BamHI released gD 
structural gene from the initial vector (pAc-gD1) and from the recombinant 
baculovirus (vAc-gD1). 
FIG. 4 is a coomassie blue staining of recombinant baculovirus infected 
cell extracts following SDS-PAGE. M. molecular weight markers; B. wild 
type baculovirus infected cells; gD.vAc-gD1. The arrows on the right 
indicate the positions of the major glycosylated gD band in gD and the 
wild type polyhedron protein visible in B. 
FIG. 5 is a Western Blot analysis of baculovirus expressed gD 
glycosylation. M. molecular weight markers; 1. Veto cells infected with 
HSV-1 at an MOI of 10 for 24 hr.; 2. vAc-gD1 infected cells at 48 hr.; 3. 
vAc-gD1 infected cells at 72 hr.; 4. Tunicamycin treated vAc-gD1 infected 
cells; 5. Endo-H treated vAc-gD1 infected cells; 6. Endo-F treated vAc-gD1 
infected cells. 
FIGS. 6A, 6B, & 6C show the immunofluorescence of recombinant 
baculovirus-infected cells. a. Recombinant vAc-gD1 infected cells, total 
fluorescence; b. vAc-gD1 infected cells, surface fluorescence; c. wild 
type baculovirus infected cells, total fluorescence.

V. DETAILED DESCRIPTION OF THE INVENTION 
The present invention utilizes recombinant DNA techniques to insert a DNA 
sequence coding for an HSV-1 glycoprotein, protein or a portion thereof, 
into a DNA vector, such that the vector is capable of replicating and 
directing expression of the glycoprotein or protein gene in a foreign 
host. The resulting recombinant DNA molecule is introduced into insect 
cells to enable high production of the glycoprotein, or protein, or a 
portion or molecular variant thereof by the host cells. The glycoprotein 
or protein produced is then isolated and purified for use in immunotherapy 
against both HSV type 1 and type 2. 
A. Preparation Of HSV-1 Glycoproteins 
It is to be understood that other procedures may be utilized and that 
changes may be made without departing from the scope of the invention. The 
following detailed description is, therefore, not to be taken in a 
limiting sense, and the scope of the present invention is best defined by 
the appended claims. 
1. Baculovirus expression of HSV-1 gD 
The DNA sequence encoding the complete herpes simplex virus type 1 (HSV-1) 
glycoprotein D (gD) was inserted into a baculovirus transfer vector under 
control of the polyhedron gene promoter of the baculovirus Autographa 
californica nuclear polyhedrosis virus (AcNPV). After co-transfection of 
Spodoptera frugiperda (Sf9) insect cells with wild-type AcNPV DNA and the 
recombinant transfer vector DNA, polyhedron-negative recombinants that 
expressed high levels of HSV-1 gD were isolated using immunoaffinity 
selection with antibody coated magnetic particles followed by plaque 
purification. These recombinant baculoviruses expressed a protein that was 
slightly smaller than virion HSV-1 gD made in Vero cells. The HSV-1 gD 
recombinant protein was expressed at high levels, was glycosylated, was 
found on the membrane of Sf9 cells, and reacted with gD specific 
antibodies. The procedure is described in detail (12) as follows: 
a. Materials and methods 
i. Viruses and cells 
The E2 strain of Autographa californica nuclear polyhedrosis virus (AcNPV) 
(baculovirus) and Spodoptera frugiperda clone 9 (Sf9) insect cells were 
gifts from Dr. Max Summers, Texas A & M. Insect cells were grown in TNM-FH 
media (13) containing 10% fetal bovine serum. Wild-type and recombinant 
baculoviruses were grown according to the procedures of Summers and Smith 
(13). Plaque purified HSV-1 (strains McKrae and KOS) and Vero cells were 
grown using standard techniques as previously described (14). 
ii. Construction of AcNPV recombinant transfer vector 
Plasmid pSS17 (HSV-1 strain KOS, a gift from D. C. Johnson, Ontario, 
Canada) was double digested with Afl II/Mae I and the 1.2 kb fragment 
containing the complete coding region of gD was isolated and blunt ended 
into the Hinc II site of pGEM3 (see FIG. 1). The construct was linearized 
with Hind III, digested briefly with Bal 31 exonuclease to remove unwanted 
nucleotides upstream from the first ATG in the coding sequence, and BamHI 
linker was added. The resulting fragment was isolated and ligated into the 
unique BamHI site of the vector pAcYM I (15) in lieu of the polyhedron 
structural gene (under control of the strong polyhedron gene promoter). 
Partial sequence analysis indicated that the cloned gD has a short 
noncoding region of 6 nucleotides at the 5' end. No HSV noncoding 
sequences are present after the termination codon (TAG) at the 3' end. 
iii. Preparation of magnetic particles for selection of recombinant gD 
Ten ml of magnetic goat-anti rabbit IgG (Advanced Magnetics Inc., 
Cambridge, Mass.) were transferred to a 75 ml tissue culture flask. A 
large magnet (supplied by the manufacturer) was placed on one flat surface 
of the flask to attract and retain the magnetic goat anti-rabbit IgG and 
the supernatant was decanted and discarded. After washing the magnetic 
material with coupling buffer (100 mM PBS pH 7.4, 0.1% BSA, 0.1% non-fat 
dry milk, and 1 mg/ml AcNPV total protein extract) 1 ml of gD polyclonal 
antibody (40 .mu.g) in 9 ml coupling buffer was added to the BioMag 
(magnetic goat anti-rabbit IgG). The mixture was agitated overnight at 
4.degree. C., and then magnetically separated. After two successive washes 
with coupling buffer and two washes with TNM-FH, the BioMag-gD antibody 
complexes were resuspended in 10 ml TNM-FH and stored at 4.degree. C. 
iv. Transfection and selection of recombinant viruses 
Sf9 cells were cotransfected with purified infectious AcNPV DNA and pAc-gD 
1 plasmid DNA essentially as described (13). After four days of incubation 
at 28.degree. C. and the appearance of CPE, the infected cells were 
screened by immunoaffinity selection. The cell pellet was harvested and 
incubated with 1 ml of anti-gD antibody-coated magnetic particles and 1 ml 
of media for 60 minutes on ice. The cell suspension was magnetically 
separated, fresh media was added to the magnetically collected cells and 
separation (washing) was repeated five times at room temperature. The 
selected material was used to infect confluent monolayers of Sf9 cells. 
Following viral growth the supernatant was titrated on confluent 
monolayers of Sf9 cells. Plaques exhibiting no evidence of occlusion 
bodies (viral polyhedra) as determined by transmission light microscopy 
were recovered and retitered on Sf9 cells to obtain recombinant, 
polyhedron-negative viruses. Following a third plaque picking, high 
titered stocks (10.sup.7 -10.sup.8 PFU/ml) of recombinant viruses were 
obtained. 
v. Preparation of viral DNA 
Sf9 cells were infected with recombinant virus at a multiplicity of 10 
PFU/cell and incubated at 28.degree. C. for 27 hours. The infected cells 
were freeze-thawed and centrifuged for 10 minutes at 1000.times.g to 
remove cell debris. The procedures used for virus isolation and viral DNA 
extraction (from the supernatant fluid) and blotting have been previously 
described (16). 
vi. Western blots 
Western immunblot analyses were carried out under denaturing conditions. 
Samples for SDS-PAGE were disrupted in electrophoresis sample buffer 
containing 2% SDS and 10% 2-mercaptoethanol and heated at 100.degree. C. 
for three minutes. Proteins were separated by SDS-PAGE (17), and 
transferred to nitrocellulose paper by electrophoresis as described by 
Towbin et al. (18). After transfer, nitrocellulose blots were blocked in 
BLOTTO (5% nonfat dry milk in PBS) and then reacted with anti-gD 
polyclonal antibody or total HSV-1 antibody 1 hour at 4.degree. C. Bound 
antibody was detected by reacting the blots with .sup.125 I-protein A for 
1 hour at 25.degree. C. followed by autoradiography. 
vii. Endoglycosidase H and endoglycosidase F 
To determine if complex sugars were added as part of the gD glycosylation 
protein, Endoglycosidase H (Endo-H) and Endoglycosidase F (Endo-F) 
treatments were done on lysed infected cells as described by the 
manufacturer (Boehringer Mannheim Biochemicals). Endo-H removes high 
mannose chains while Endo-F removes both high mannose and hybrid sugars. 
viii. Tunicamycin treatment 
To determine if the expressed gD underwent N-glycosylation, infected cell 
monolayers were treated with 4 .mu.g/ml tunicamycin (an inhibitor of 
asparagine-linked glycosylation) in TNM-FH media for 48 hours as described 
(19). 
ix. Immunofluorescence 
Sf9 cells were infected with wild-type AcNPV or recombinant baculoviruses 
expressing gD at a multiplicity of infection of 10 PFU/cell and incubated 
for 72 hours. To look at total fluorescence, cells were washed with PBS, 
fixed with acetone and anti-gD polyclonal antibody (provided by Dr. 
Richard Eberle) was added and incubated for 1 hour at 37.degree. C. 
Alternatively, to determine cell surface immunofluorescence, unfixed, 
unpermeabilized cells were washed with PBS and incubated with antibody for 
1 hour at 4.degree. C. After washing, slides were fixed with acetone. 
Slides for total and surface fluorescence were then washed with PBS, 
stained with fluorescein-conjugated goat anti-rabbit IgG antibody for 1 
hour at 37.degree. C., washed again with PBS, and examined for 
fluorescence. 
2. Results 
a. Construction of recombinant viruses expressing gD 
The strategy for the construction of the baculovirus transfer vector 
containing the complete gD open reading frame from HSV-1 is shown in FIG. 
1. A complete DNA copy of the gD gene from the BamHI J fragment, was 
isolated by restriction enzyme digestion with AflII/MaeI. Most of the 5' 
noncoding sequences were removed by Bal31 digestion. The resulting DNA was 
then inserted into the BamHI site of the pAcYM1 vector (FIG. 1). As 
confirmed by restriction enzyme analysis and partial sequencing, this 
construct contains the entire sequence of the gD gene. It has a non-coding 
region of only 6 nucleotides in front of the first ATG. This is followed 
by the complete coding region of 1182 nucleotides. To transfer the gD gene 
into the baculovirus AcNPV genome, Sf9 cells were cotransfected with 
pAc-gD1 DNA and infectious AcNPV DNA. Putative recombinant viruses were 
enriched by immunoaffinity selection, which was followed by three cycles 
of polyhedron-negative plaque purification. In this study, one round of 
immunoselection increased the efficiency of obtaining recombinant viruses 
by several fold with yields of better than 8% recombinants in the first 
plaque purification cycle. 
b. Western blot analysis 
Confluent monolayers of Sf9 cells were infected at a multiplicity of 10 
PFU/cell with 11 individual recombinant baculoviruses obtained after three 
plaque purifications and total protein extracts were analyzed by Western 
blotting. Our vAc-gD1 recombinants produced 6 protein bands that reacted 
with both total HSV-1 polyclonal antibody (FIG. 2) and gD polyclonal 
antibody (FIG. 5). A band with an apparent molecular weight of 43 kDa 
corresponds to the non-glycosylated primary gD polypeptide that has a 
predicted molecular weight of 43,291 Da. Two larger bands (50 kDa and 52 
kDa) ran as a very tight doublet that was not resolved in this blot. These 
two bands presumably represent the partially glycosylated precursor pgD 
and mature gD (20, 21) respectively. 
The three smallest bands had apparent molecular weights of 33, 24, and 22 
kDa. A similar pattern of bands was obtained with immunoaffinity column 
(Affi-Gel 10; Bio-Rad) purified gD (using gD monoclonal antibody, a gift 
from Dr. D. Wiley). 
Western blot analysis of recombinant infected cell medium did not detect 
any gD. This suggests that the expressed gD is retained in the cell or in 
the cell membrane as with HSV-1 infected cells and is not secreted into 
the medium. 
c. Southern blot analysis 
Since no obvious differences in the expression levels or the sizes of any 
of the gD related bands were seen among the 11 recombinant viruses, one 
recombinant virus was arbitrarily selected for subsequent study and 
designated vAc-gD1. To verify the presence of full length HSV-1 gD DNA in 
vAc-gD1, the baculovirus DNA was digested with the restriction enzyme 
BamHI and Southern blots were done using the gD gene as a probe (FIG. 3). 
As can be seen by both ethidium bromide staining of the DNA (FIG. 3a) and 
Southern analysis using a gD specific probe (FIG. 3b), BamHI digestion of 
the gD recombinant generated a band of the expected size (approximately 
1.2 kb). This corresponds to the HSV-1 gD gene cloned into the expression 
vector (FIG. 3). 
d. Visualization of expressed gD by SDS-PAGE and coomassie blue staining 
Total cell extracts from wild type baculovirus and vAc-gD1 recombinant 
baculovirus infected cells were run on SDS-PAGE and protein bands were 
stained with coomassie blue. The polyhedron protein band seen in wild type 
baculovirus infected cells (FIG. 4, lane B) was missing in vAc-gD1 
infected cells, while a new, larger band of similar intensity was present 
in the vAc-gD1 infected cells (FIG. 4, lane gD). Neither band was seen in 
uninfected cells. This new recombinant band had an apparent molecular 
weight of approximately 50-52 kDa, corresponding in size to the tight 
doublet upper bands seen by Western analysis (FIG. 2, bands 50 and 52 
kDa), and represented the major expressed gD species in this recombinant 
baculovirus. 
Visual observation of the stained gel suggested that the amount of 
expressed gD was similar to the amount of polyhedron in wild type 
baculovirus infected cells (FIG. 4, compare gD to P in lanes gD and B). To 
confirm this and to more accurately estimate the relative expression level 
of gD, the coomassie blue stained gel shown in FIG. 4 was scanned on a 
laser densitometer. The area under the combined peak representing gD and 
pgD was similar to the area under the peak for the polyhedron protein in 
wild type baculovirus infected cells. Identical results were obtained in 
scans of additional gels. Since the polyhedron protein has been estimated 
to comprise up to 40% of total cellular protein, this analysis indicates 
that the recombinant gD is expressed at very high levels (22). 
e. Glycosylation of gD 
To confirm that the expressed gD underwent glycosylation, tunicamycin 
treatment was done to prevent N-glycosylation in infected Sf9 cells. 
Infected cells were treated with 4 .mu.g tunicamycin/ml of TNM-FH media 
from 0-48 h post infection, and total cell extracts were analyzed by 
Western blots using polyclonal anti-gD antibody. The tunicamycin treatment 
(FIG. 5, lane 4) reduced the apparent size of gD relative to the control 
(lanes 2 and 3). This result indicates that like native gD, the untreated 
expressed gD was glycosylated and contained N-linked sugars. 
To determine if the expressed gD contained complex sugars, vAc-gD1 infected 
cell lysates were treated with Endoglycosidase-H (Endo-H, removes high 
mannose sugars) or Endoglycosidase-F (Endo-F, removes high mannose and 
hybrid sugars). As seen in FIG. 5 lane 5, gD was partially resistant to 
digestion by Endo-H. In contrast, Endo-F digestion decreased the apparent 
molecular weight of gD to approximately 45 kDa (FIG. 5, lane 6). Thus, 
similar to native gD, the expressed gD was partially resistant to Endo-H 
and susceptible to Endo-F. Therefore, like native gD the recombinant gD 
was glycosylated and contained N-linked hybrid sugars. 
f. Immunofluorescence of recombinant gD in insect cells 
To determine whether the expressed gD was transported to the cell surface, 
vAc-gD1 infected Sf9 cells were examined by indirect immunofluorescent 
antibody staining using polyclonal antibody to gD. Immunofluorescence was 
readily observed in recombinant-infected cells (FIG. 6a). No 
immunofluorescence was seen in cells infected with AcNPV (FIG. 6c) or in 
uninfected Sf9 cells. To look specifically for gD on the cell surface, 
indirect immunofluorescent antibody staining was done on cells prior to 
fixation (and permeabilization) (FIG. 6b). The surface fluorescence on 
vAc-gD1 infected cells was strong and comparable to that observed for 
permeabilized fixed cells. This indicates that the expressed gD was 
correctly transported to and anchored in the cell surface. 
3. Baculovirus expression of HSV-1 gB, gC, gE, gG, gH or gI 
Glycoproteins gB, gC, gE, gG, gH and gI (McGeoch et al., Journal of General 
Virology 69:1531-1574(1988)) were similarly expressed in baculovirus 
(23-28). In all cases, high level expression of a protein similar in size 
to the native glycoprotein was obtained. All the expressed glycoproteins 
were glycosylated, reacted with the appropriate monoclonal or polyclonal 
antisera, and were correctly transported to the cell surface. 
B. Vaccination 
1. Outline of Vaccine Trial 
Rabbits. New Zealand White (NZW) male rabbits (approximately 2 kg each) are 
used for all experiments. As discussed above, these animals develop a 
primary and recurrent ocular herpetic disease that mimics HSV keratitis in 
man. 
Virus and vaccines. McKrae, a highly pathogenic stromal disease causing 
HSV-1 strain, with a very high spontaneous reactivation rate is used for 
establishment of latency. Strain RE or W will be used in some experiments 
to confirm that results are not strain specific. 
Day 0: Ocular infection for establishment of latency. Rabbits are 
bilaterally infected by placing 2.times.10.sup.5 PFU (McKrae) into the 
conjunctival cul-de-sac, closing the eye, and rubbing gently for 30 
seconds. Although this dose of virus results in the death of approximately 
30% of the rabbits, this is the lowest dose at which all survivors become 
latently infected in both eyes (really both trigeminal ganglia). Although 
latency cannot be judged at this point without the loss of the rabbit for 
the vaccine experiment, our extensive experience indicates that virtually 
every trigeminal ganglia harbors HSV-1 latency under these conditions. At 
the termination of the vaccine trial, the trigeminal ganglia from those 
eyes that have not shown spontaneous or induced reactivation will be 
removed and explant co-cultivation done to confirm that latency had been 
established. In our experience this strain and dose combination results in 
the highest number of known latently infected and spontaneously 
reactivating eyes, (starting with a given number of rabbits) thus using 
the fewest possible rabbits. Also with this regimen, no more than 1% of 
rabbits become blind in both eyes (these rabbits are immediately 
sacrificed). 
Subconjunctival vaccination is done using topical proparacaine for 
anesthesia prior to subconjunctival injection of 0.2-0.5 ml of vaccine 
given with an insulin syringe and a small gauge needle. The injection is 
given in either the upper or lower cul de sac making an effort that the 
vaccine material remains subconjunctival and does not leak out. 
Enteric vaccination is done by gavage down the rabbits throat. 
Vaccination by surface drops and collagen shield is as described above. 
Glycoprotein purification. Glycoproteins will be purified by differential 
centrifugation of freeze-thawed or detergent disrupted cell extracts, 
followed by immunoaffinity purification through columns with covalently 
bound HSV-1 antibody. 
ISCONs with Quil-A are made as previously described in the literature (29, 
30). 
Day 21: Latency is considered to be established in all survivors (see 
Specific methods below for a discussion). Rabbits are randomly divided 
into trial and control groups and vaccinated ocularly. 
Day 35: Vaccination repeated. 
Day 49-118: Tear films are collected once a day, 5 days a week to look for 
spontaneous shedding indicative of HSV reactivation. 
Day 49-118: Eyes are examined 3.times./week (prior to tear film cultures) 
by slit lamp biomicroscopy to directly monitor epithelial keratitis, 
stromal disease and scarring. 
Ocular parameters. Severity of ocular disease is scored on a 0 to 4 scale 
in a masked fashion by examination with slit lamp biomicroscopy using 1% 
methylene blue to delineate epithelial ulceration. Iritis and stromal 
keratitis are also scored on a 0 to 4 scale. 
In vivo reactivation is done by iontophoresis with 6-hydroxydopamine 
followed by topical epinephrine (31). 
Blood and tear samples are taken for immunological analysis prior to 
initial infection (day 1), prior to vaccinations (days 20 and 34), 2 weeks 
after the second vaccination (day 49), and at the end of the experiment 
(&gt;day 118). 
In vivo reactivation. Co-cultivation of trigeminal ganglia is done as 
previously described (32). 
Serum neutralizing antibody titers are done by plaque reduction assays 
(33). 
Local ocular sIgA and IgG titers are done using tears collected on a "sno 
strip" (34) for human sIgA cervical HSV-2, with adjustments made to detect 
rabbit (rather than human) HSV-1 (rather than HSV-2) specific sIgA and 
IgG. Correlations between IgG and IgA neutralization titers and ELISAs 
will be done. 
Systemic IgA and IgG titers are done from sera by ELISA (35). 
Lymphocyte proliferation responses will be monitored by checking for T 
cells that will proliferate upon stimulation with HSV proteins (36). 
Peripheral blood mononuclear cells (PBMC) are collected from latently 
infected, latently infected immunized, and control rabbits by venipuncture 
into preservative-free heparinized syringes, with subsequent purification 
by Ficoll-hypaque centrifugation. PBMC are resuspended to a concentration 
of 2.5.times.10.sup.5 cells/ml in RPMI-1640 medium containing 15% 
heat-inactivated fetal bovine serum and antibiotics (RPMI-15% FBS). 200 
.mu.l of PBMC (5.times.10.sup.4) is added to each of four replicate round 
bottom microtiter plate wells, followed by the addition of 100 .mu.l of 
1.0 or 5.0 .mu.g/ml solutions of expressed HSV-1 glycoprotein, 100 .mu.l 
of UV-light inactivated HSV-1 (10.sup.6 PFU/ml prior to UV irradiation), 
or 100 .mu.l of RPMI-15% PBS as a control. Seven days after the onset of 
stimulation, 1 .mu.Ci of .sup.3 H!thymidine (.sup.3 H!TdR, NEN, Boston, 
Mass.) is added for the last 6 hours of incubation at 37.degree. C. Cells 
are then harvested by using a multiple well harvesting device, and .sup.3 
H!TdR incorporation determined by liquid scintillation counting. 
Therapeutic effectiveness is determined by comparing ocular shedding, 
recurrent epithelial keratitis, recurrent stromal keratitis, and scarring 
to mock vaccinated controls. All animal work and analyses will be masked 
to eliminate bias. 
2. Controls 
All vaccine trials were compared to a mock vaccine for the amount of ocular 
shedding, the amount of recurrent stromal disease and the levels of immune 
response. 
Spontaneous Ocular Shedding as a Valid Predictor of Corneal Lesions 
Corneal epithelial lesions and stromal scarring are usually preceded by 
detectable levels of ocular HSV shedding. Hence, shedding is almost 
certainly a prerequisite for recurrent epithelial and stromal lesions. All 
else being equal, it was surmised that a decrease in shedding, i.e., less 
culture-detectable infectious virus in tears, will result in decreased 
corneal disease. Ocular shedding was therefore determined by collecting 
tear films once a day, five days per week from each eye and culturing them 
for infectious virus between day 49 (two weeks after the final 
vaccination) and day 118. 
4. Induced Ocular Shedding 
Induced ocular shedding can be accomplished by iontophoresis with 
6-hydroxydopamine followed by topical epinephrine as described previously 
in the literature. 
5. Selection of Vaccines 
Various combinations and permutations of the seven HSV-1 glycoproteins or 
proteins can be made and used. Among these combinations include HSV-1 gD 
and gB, a combination of all seven HSV-1 glycoproteins in equimolar 
amounts, and any combination of one or more of the seven HSV-1 
glycoproteins and proteins. 
6. Adjuvants 
MTP-PE is the adjuvant of choice for subconjunctival vaccinations. MTP-PE 
may be administered with the HSV-1 glycoproteins alone or in combination 
with other adjuvants. MTP-PE may also be encapsulated in a liposome in 
combination with one or more HSV-1 glycoproteins or proteins. The use of 
other adjuvants, known or yet to be discovered, however, is not foreclosed 
by the disclosure of MTP-PE as the preferred adjuvant. 
For systemic vaccination, i.e. intramuscular (IM) or subcutaneous (SC) 
vaccination, the most powerful adjuvants may be used. These include, but 
are not limited to, alum, Freund's complete, Freund's incomplete, MTP-PE, 
ISCOMs (Quil A), and any combination of these. 
EXAMPLE 1 
Systemic Therapeutic Vaccination with recombinant HSV-2 gB-gD 
Methods 
Thirty-six NZW rabbits with culture proven binocular HSV-1 McKrae infection 
were given subcutaneous vaccinations (0.5 ml) of Chiron gB2 and gD2 with 
MTP-PE adjuvant on days 21 and 39 post infection. The mock vaccinated 
group received tissue culture media pursuant to the same schedule. Daily 7 
day/wk swab cultures (primary rabbit kidney) were taken for 36 days 
beginning 3 weeks after the second vaccination. All positive cultures were 
confirmed by neutralization. ELISA titers (capture Ag purified KOS) were 
done on blood samples taken 5 weeks after the second vaccination (sera 
were diluted in 1/2 log steps from 1/100 to 1/300,000). Daily 5 day/wk 
slit lamp biomicroscopy was done over the same period. 
Results 
TABLE 1 
______________________________________ 
Spontaneous ocular shedding and dendritic 
positive eyes, following systemic vaccination. 
# Ani- 
Vaccine & mals/ % Pos- 
# Den- % Den- 
Adjuvant 
Route # Eyes + Culture 
itive dritic dritic 
______________________________________ 
Chiron Subcu- 19/38 124/1368 
9.6% 121/1140 
1.93% 
gB2 + taneous 
gD2 with 
MTP-PE 
Mock Subcu- 17/34 151/1224 
12.34% 
19/1020 
1.86% 
without 
adjuvant 
______________________________________ 
*all eyes cultured for 36 consecutive days 
**slit lamp biomicroscopy (5 days/wk) for 30 days 
As illustrated in Table 1, there is no significant difference in the number 
of positive cultures or the number of eyes with dendritic figures between 
the mock and the Chiron vaccinated groups. There is also no difference in 
(data not shown) (1) the number of eyes that have 1 or multiple 
recurrences; (2) the number of rabbits that have 1 or multiple 
recurrences; (3) the average duration of shedding events; (4) the severity 
of keratitis; or (5) the HSV-1 ELISA titer in high vs. low recurrence 
animals, although the average titer is slightly higher in the Chiron than 
the mock vaccinated group. There was no difference in long term scarring 
and no signs of ocular toxicity. 
Although there was no increase in shedding or dendritic keratitis produced 
by systemic vaccination with gB2+gD2 with MTP-PE, neither was any 
significant decrease seen in the same parameters with the same systemic 
subunit vaccine. Consequently, systemic vaccination does not produce 
adequate ocular protection from HSV-1 recurrences. 
EXAMPLE 2 
Systemic Vaccination with HSV-1 gD 
Methods 
V52, a genetically engineered vaccine virus recombinant that expresses the 
HSV-1 glycoprotein gD (obtained from B. Moss) was used to vaccinate 
rabbits intradermally. It should be noted, however, that the HSV-1 gD may 
also be obtained from the procedures described above or any equivalent. 
Significant HSV-1 neutralizing antibody titers were produced, although 
they were not as high as those induced by vaccination with live HSV-1. The 
mock vaccinated group received a vaccinia-influenza recombinant, V36. The 
positive control group received live attenuated HSV-1 virus (KOS) 
subcutaneously. Rabbits were challenged ocularly with topical application 
of 2.times.10.sup.5 PFU of HSV-1 McKrae. Eyes were monitored for 35 days 
for epithelial keratitis, stromal keratitis, and iritis. 
Results 
V52 gD provided a small amount of protection against HSV-1 induced 
epithelial keratitis (p=0.02) and long term stromal scarring (p=0.04). In 
addition, 89% of the V36 vaccinated rabbits (negative control) could be 
induced to reactivate. In contrast, only 55% of the V52 vaccinated rabbits 
could be induced to reactivate, suggesting a modest protective effect. The 
positive control rabbits vaccinated subcutaneously with KOS produced the 
same results as seen with gD. Hence, systemic vaccination with HSV-1 gD on 
live attenuated KOS HSV-1 did not produce adequate ocular protection from 
HSV-1 recurrences. 
EXAMPLE 3 
Local ocular vaccination with KOS 
Methods 
Twelve NZW rabbits were vaccinated with the non-pathogenic live HSV-1 
strain, KOS. KOS replicates very well in the eye, however, even at 
extremely high titers, the KOS strain causes minimal eye disease in the 
rabbit. The rabbits were vaccinated by placing 5.times.10.sup.8 virus in 
each eye and holding the eye closed for 30 seconds. A control group of 12 
rabbits were each mock infected with tissue culture media. Four weeks 
later, the rabbits were challenged with HSV-1 McKrae and the eyes were 
observed by slit lamp biomicroscopy over a 2 week period. The severity of 
disease was scored on a scale of 0-4. 
Results 
Maximum eye involvement occurred on day 7, the average severity of disease 
is shown in Table 2. 
TABLE 2 
______________________________________ 
Ocular vaccination protects against primary ocular infection. 
EYE EYE SYSTEMIC 
Mock KOS McKrae 
vaccinated 
vaccinated 
Vaccinated 
______________________________________ 
Epithelial keratitis 
1.4+/-0.5 0.0 2.1+/-1.2 
Stromal keratitis 
2.0+/-0.5 0.0 1.9+/-1.0 
Iritis 2.1+/-1.7 0.1+/-0.05 
1.5+/-1.4 
______________________________________ 
*McKrae data is from the V52 experiment described above. 
As demonstrated above, the protection afforded against ocular challenge at 
4 weeks was almost complete. Ocular vaccination was dramatically more 
efficient than the systemic vaccination demonstrated in Examples 1 and 2 
above. This experiment therefore suggests that a local ocular vaccine may 
afford the necessary protection in the rabbit against ocular challenge 
that is lacking with systemic vaccination. Moreover, since ocular 
vaccination appears more powerful in protecting against primary ocular 
infection, it follows then that it is also likely to be more powerful in 
protecting against ocular recurrence. Because the rabbit ocular model of 
HSV infection mimics the human infection, local ocular vaccination also 
appears to represent the best protection from HSV-1 ocular recurrences in 
humans. 
EXAMPLE 4 
Local Ocular Vaccination with Expressed HSV-1 gB and gD 
Methods 
Five NZW rabbits with culture proven binocular HSV-1 McKrae infection were 
given subconjunctival vaccinations on days 32 and 54 post infection. The 
vaccine comprised equal amounts of expressed gB1 and gD1 mixed 50/50 with 
MTP-PE adjuvant. Nine eyes were vaccinated: 4 animals bilateral, 1 
unilateral. Daily 7 day/wk swab cultures (primary rabbit kidney) were 
taken for 22 days beginning 3 weeks after the second vaccination. All 
positive cultures were confirmed by neutralization. Daily 5 day/wk slit 
lamp biomicroscopy was carried out over the same period. 
Results 
There were no corneal or anterior chamber abnormalities seen on 
biomicroscopy that differed from the mock vaccinated animals. All eyes 
showed mild generalized infection for 1 week following subconjunctival 
vaccination. Three eyes showed sustained localized mild conjunctival 
infection at the vaccination site. 
The results shown in Table 3 below are intriguing and suggest reduced 
shedding following ocular subconjunctival vaccination with gB1+gD1. These 
experiments demonstrate that (1) local conjunctival vaccination is 
possible without apparent harm to the recipients; and (2) local ocular 
vaccination is more effective then systemic vaccination in preventing HSV 
ocular recurrences. 
TABLE 3 
______________________________________ 
Spontaneous ocular shedding and dendritic keratitis following local 
subconjunctival vaccination with baculovirus expressed 
HSV-1 gB and gD. 
# Ani- 
Vaccine & mals/ + Cul- 
% Pos- 
# Den- 
% Den- 
Adjuvant 
Route # Eyes ture itive dritic 
dritic 
______________________________________ 
gB1 + gD1 
Local 5/9 9/198 
4.55% 
2/126 
1/59% 
with subcon- 
MTP-PE junc- 
tival 
KOS with- 
Systemic 17/34 75/748 
10.02% 
11/476 
2.31% 
out suncu- 
Adjuvant 
taneous 
______________________________________ 
*all eyes cultured for 22 consecutive daysKOS numbers are reported for th 
days that the subconjunctival animals were cultured, i.e. Days 77-99 post 
infection. 
**slit lamp biomicroscopy (5 days/wk) for 14 days 
In summary, the Examples above illustrate that: 1) systemic vaccination 
with HSV-2 gB+gD with MTP-PE did not produce adequate ocular protection 
from HSV-1 recurrences; 2) systemic vaccination with HSV-1 gD or live 
attenuated KOS HSV-1 also did not produce adequate ocular protection from 
HSV-1 recurrences; 3) local ocular vaccination with a live nonpathogenic 
HSV-1 strain did protect against primary ocular infection; and 4) local 
ocular vaccination with HSV-1 gB and gD was effective in preventing HSV 
ocular recurrences. These examples illustrate that a local ocular 
immunogenic comprised of one or more HSV-1 glycoproteins or proteins, 
would greatly alleviate HSV ocular recurrences, the most frequent serious 
viral eye infection in humans in the United States and a major cause of 
viral induced blindness in the world. 
The foregoing detailed description is given for clearness of understanding 
only, and no unnecessary limitations are to be understood or inferred 
therefrom, as modifications within the scope of the invention will be 
obvious to those skilled in the art. 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1204 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TATAAATACGGATCCTCCGGTATGGGGGGGGCTGCCGCCAGGTTGGGGGCCGTGATTTTG60 
TTTGTCGTCATAGTGGGCCTCCATGGGGTCCGCAGCAAATATGCCTTGGTGGATGCCTCT120 
CTCAAGATGGCCGACCCCAATCGCTTTCGCGGCAAAGACCTTCCGGTCCTGGACCAGCTG180 
ACCGACCCTCCGGGGGTCCGGCGCGTGTACCACATCCAGGCGGGCCTACCGGACCCGTTC240 
CAGCCCCCCAGCCTCCCGATCACGGTTTACTACGCCGTGTTGGAGCGCGCCTGCCGCAGC300 
GTGCTCCTAAACGCACCGTCGGAGGCCCCCCAGATTGTCCGCGGGGCCTCCGAAGACGTC360 
CGGAAACAACCCTACAACCTGACCATCGCTTGGTTTCGGATGGGAGGCAACTGTGCTATC420 
CCCATCACGGTCATGGAGTACACCGAATGCTCCTACAACAAGTCTCTGGGGGCCTGTCCC480 
ATCCGAACGCAGCCCCGCTGGAACTACTATGACAGCTTCAGCGCCGTCAGCGAGGATAAC540 
CTGGGGTTCCTGATGCACGCCCCCGCGTTTGAGACCGCCGGCACGTACCTGCGGCTCGTG600 
AAGATAAACGACTGGACGGAGATTACACAGTTTATCCTGGAGCACCGAGCCAAGGGCTCC660 
TGTAAGTACGCCCTCCCGCTGCGCATCCCCCCGTCAGCCTGCCTCTCCCCCCAGGCCTAC720 
CAGCAGGGGGTGACGGTGGACAGCATCGGGATGCTGCCCCGCTTCATCCCCGAGAACCAG780 
CGCACCGTCGCCGTATACAGCTTGAAGATCGCCGGGTGGCACGGGCCCAAGGCCCCATAC840 
ACGAGCACCCTGCTGCCCCCGGAGCTGTCCGAGACCCCCAACGCCACGCAGCCAGAACTC900 
GCCCCGGAAGACCCCGAGGATTCGGCCCTCTTGGAGGACCCCGTGGGGACGGTGGCGCCG960 
CAAATCCCACCAAACTGGCACATACCGTCGATCCAGGACGCCGCGACGCCTTACCATCCC1020 
CCGGCCACCCCGAACAACATGGGCCTGATCGCCGGCGCGGTGGGCGGCAGTCTCCTGGCA1080 
GCCCTGGTCATTTGCGGAATTGTGTACTGGATGCGCCGCCACACTCAAAAAGCCCCAAAG1140 
CGCATACGCCTCCCCCACATCCGGGAAGACGACCAGCCGTCCTCGCACCAGCCCTTGTTT1200 
TACT1204 
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