U.S. patent application number 10/370200 was filed with the patent office on 2003-08-14 for soluble herpesvirus glycoprotein complex vaccine.
This patent application is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to Cohen, Gary H., Dubin, Gary, Eisenberg, Roselyn J., Peng, Tao.
Application Number | 20030152583 10/370200 |
Document ID | / |
Family ID | 26960306 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030152583 |
Kind Code |
A1 |
Cohen, Gary H. ; et
al. |
August 14, 2003 |
Soluble herpesvirus glycoprotein complex vaccine
Abstract
The invention is directed to a herpes simplex virus vaccine
comprising a herpes simplex virus glycoprotein H-glycoprotein L
complex. The invention is also directed to a vaccine comprising a
DNA encoding a herpes simplex virus glycoprotein H-glycoprotein L
complex. Also included is an antibody which specifically binds to a
herpes simplex virus glycoprotein H-glycoprotein L complex and DNA
encoding the same.
Inventors: |
Cohen, Gary H.; (Havertown,
PA) ; Eisenberg, Roselyn J.; (Haddonfield, NJ)
; Peng, Tao; (San Diego, CA) ; Dubin, Gary;
(La Hulpe, BE) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP
1701 MARKET STREET
PHILADELPHIA
PA
19103-2921
US
|
Assignee: |
The Trustees of the University of
Pennsylvania
|
Family ID: |
26960306 |
Appl. No.: |
10/370200 |
Filed: |
February 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10370200 |
Feb 20, 2003 |
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09658056 |
Sep 8, 2000 |
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6541459 |
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09658056 |
Sep 8, 2000 |
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08904484 |
Jul 31, 1997 |
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6156319 |
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08904484 |
Jul 31, 1997 |
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08280442 |
Jul 25, 1994 |
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5807557 |
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Current U.S.
Class: |
424/186.1 ;
424/193.1; 435/455; 435/69.3; 514/44R |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 48/00 20130101; C12N 2710/16622 20130101; A61K 39/00 20130101;
A61P 31/22 20180101; C07K 14/005 20130101 |
Class at
Publication: |
424/186.1 ;
514/44; 424/193.1; 435/455; 435/69.3 |
International
Class: |
A61K 031/70; A01N
043/04; C12N 015/09; A61K 039/12; A61K 039/385; C12N 015/63; C12N
015/85; C12N 015/87 |
Goverment Interests
[0002] Portions of this invention were made using funds from the
U.S. Government (Public Service Health Grant Nos. NS-30606 from the
National Institute of Neurological Diseases and Stroke, AI-18289
from the National Institute of Allergy and Infectious Diseases, and
DE-08239 from the National Institute of Dental Research) and the
U.S. Government may therefore have certain rights in the invention.
Claims
What is claimed is:
1. A vaccine comprising a soluble herpes simplex virus gHt-gL
complex suspended in a pharmaceutically acceptable carrier.
2. The vaccine of claim 1, wherein said herpes simplex virus is
selected from the group consisting of herpes simplex virus type 1
and herpes simplex virus type 2.
3. The vaccine of claim 1, wherein said herpes simplex virus is
herpes simplex virus type 1.
4. The vaccine of claim 1, wherein said gHt is from a herpes
simplex virus selected from herpes simplex virus type 1 and herpes
simplex virus type 2.
5. The vaccine of claim 1, wherein said gL is from a herpes simplex
virus selected from herpes simplex virus type 1 and herpes simplex
virus type 2.
6. The vaccine of claim 1, wherein said gHt is herpes simplex virus
type 1 and comprising amino acid residues selected from the group
consisting of 1-792, 1-648, 1-475, 1-324 and 1-275.
7. The vaccine of claim 1, wherein said gHt is herpes simplex virus
type 1 gHt comprising amino acids 1-324.
8. The vaccine of claim 1, wherein said herpes simplex virus is
herpes simplex virus type 1, said gHt comprises amino acids 1-792
and said gL comprises amino acids 1-168.
9. The vaccine of claim 1, further comprising a substantially pure
preparation of at least one of a herpes simplex virus gD, gB, or
gC.
10. A vaccine comprising an isolated DNA encoding a soluble herpes
simplex virus gHt-gL complex suspended in a pharmaceutically
acceptable carrier.
11. The vaccine of claim 10, further comprising an isolated DNA
encoding at least one of a herpes simplex virus gD, gB or gC.
12. A herpes simplex virus neutralizing antibody which specifically
binds to a soluble herpes simplex virus gHt-gL complex.
13. An isolated DNA encoding a herpes simplex virus neutralizing
antibody which specifically binds to a soluble herpes simplex virus
gHt-gL complex.
14. A method of immunizing a human patient against a herpes simplex
virus infection comprising administering to said patient the
vaccine of claim 1.
15. A method of immunizing a human patient against a herpes simplex
virus infection comprising administering to said patient the
vaccine of claim 9.
16. A method of immunizing a human patient against a herpes simplex
virus infection comprising administering to said patient the
vaccine of claim 10.
17. A method of immunizing a human patient against a herpes simplex
virus infection comprising administering to said patient the
vaccine of claim 11.
18. A method of treating a herpes simplex virus infection in a
human patient comprising administering to said patient the vaccine
of claim 1.
19. A method of treating a herpes simplex virus infection in a
human patient comprising administering to said patient the vaccine
of claim 9.
20. A method of treating a herpes simplex virus infection in a
human patient comprising administering to said patient the vaccine
of claim 10.
21. A method of treating a herpes simplex virus infection in a
human patient comprising administering to said patient the vaccine
of claim 11.
22. A method of treating a herpes simplex virus infection in a
human patient comprising administering to said patient the herpes
simplex virus neutralizing antibody of claim 12 suspended in a
pharmaceutically acceptable carrier.
23. A method of treating a herpes simplex virus infection in a
human patient comprising administering to said patient the isolated
DNA of claim 13 suspended in a pharmaceutically acceptable
carrier.
24. A preparation of a soluble herpes simplex virus gHt-gL
complex.
25. A substantially pure preparation of a soluble herpes simplex
virus gHt-gL complex.
26. The complex of claim 24, wherein said herpes simplex virus is
selected from the group consisting of herpes simplex virus type 1
and herpes simplex virus type 2.
27. The complex of claim 26, wherein said herpes simplex virus is
herpes simplex virus type 1.
28. The complex of claim 24, said complex being suspended in a
pharmaceutically acceptable carrier.
29. The complex of claim 27, wherein said gHt comprises amino acid
residues selected from the group consisting of 1-792, 1-648, 1-475,
1-324 and 1-275.
30. The complex of claim 27, wherein said gHt comprises amino acids
1-324.
31. The complex of claim 27, wherein said gHt comprises amino acids
1-792 and said gL comprises amino acids 1-168.
32. A cell, the DNA of said cell encoding a soluble herpes simplex
virus gHt-gL complex.
33. The cell of claim 32, wherein said cell is HL-7.
34. A method of modifying a cell to render it capable of secreting
a soluble herpes simplex virus gHt-gL complex comprising
introducing into said cell DNA encoding a truncated form of herpes
simplex virus gH being gHt and full length herpes simplex virus gL,
wherein said gHt and said gL are expressed in and secreted from
said cell.
35. The method of claim 34, wherein said gHt comprises herpes
simplex virus type 1 gH comprising amino acid residues selected
from the group consisting of 1-792, 1-648, 1-475, 1-324 and
1-275.
36. An isolated DNA comprising DNA encoding a herpes simplex virus
gHt and a substantially full length herpes simplex virus gL.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/280,442, filed on Jul. 25, 1994.
FIELD OF THE INVENTION
[0003] This invention is directed to herpesvirus vaccines.
BACKGROUND OF THE INVENTION
[0004] Herpesviruses are ubiquitous viruses which are the causative
agents of numerous diseases in both humans and animals. These
viruses are enveloped double stranded icosahedral DNA containing
viruses, which envelope is acquired by budding of the nucleocapsid
through the inner nuclear membrane. Members of the herpesvirus
family which are important human pathogens include herpes simplex
virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2),
varicella zoster virus (VZV), Epstein Barr virus (EBV),
cytomegalovirus (CMV), and human herpesviruses type 6, type 7 and
type 8 (HHV-6, HHV-7 and HHV-8).
[0005] The genome of HSV-1 encodes several glycoproteins which are
important for viral pathogenesis. Four glycoproteins glycoprotein B
(gB), glycoprotein D (gD), glycoprotein H (gH) and glycoprotein L
(gL) are essential for virus infectivity in cells in culture and
each appears to play a role in the mechanism by which the virus
enters cells (Roop et al., 1993, J. Virol. 67:2285). Glycoprotein H
is a 110 kDa protein encoded by the UL22 open reading frame of
HSV-1 (Gompels and Minson, 1986, Virology 153:230). When gH is
expressed in mammalian cell systems in the absence of other HSV-1
proteins it remains within the cell as an incompletely processed
molecule (Foa-Tomasi et al., 1991, Virology 180:474; Roberts et
al., 1991, Virology 184:609). When gH is expressed in cells which
also express gL, gH and gL form a stable complex wherein fully
processed gH is evident (Hutchinson et al., 1992, J. Virol.
66:2240). In addition, cells infected with a gL-negative mutant
produce virus particles which lack both gH and gL (Roop et al.,
1993, J. Virol. 67:2285). However, since transport of gH to the
surface of cells is reported to occur in the absence of gL, gL may
not be required in some systems for correct processing and
transport of gH (Ghiasi et al., 1991, Virology 185:187).
[0006] A recombinant vaccinia virus expressing both gH and. gL has
been used to examine whether the gH-gL complex was capable of
eliciting a protective immune response in mice. Weak levels of
HSV-1 specific neutralizing antibody were evident in mice
containing the complex and virus clearance from the site of
challenge was only marginally enhanced when the gH-gL complex was
administered to the animals compared with administration of gH
alone (Browne et al., 1993, J. Gen. Virol. 74:2813).
[0007] Currently, there are no effective herpesvirus vaccines
available for immunization of humans against any of the plethora of
diseases caused by these pathogens, although subunit preparations
comprising glycoprotein B, glycoprotein D, either alone or in
combination are currently in clinical trials. Since herpesviruses
cause recurrent and frequently fatal or permanently debilitating
infections in humans and in other animals7 there is a long felt
need for such vaccines.
SUMMARY OF THE INVENTION
[0008] The invention relates to a vaccine comprising a soluble
herpes simplex virus gHt-gL complex suspended in a pharmaceutically
acceptable carrier.
[0009] In one aspect, the herpes simplex virus is selected from the
group consisting of herpes simplex virus type 1 and herpes simplex
virus type 2. Preferably, the herpes simplex virus is herpes
simplex virus type 1.
[0010] In one embodiment, gHt is from a herpes simplex virus
selected from herpes simplex virus type 1 and herpes simplex virus
type 2.
[0011] In another embodiment, the gL is from a herpes simplex virus
selected from herpes simplex virus type 1 and herpes simplex virus
type 2.
[0012] In yet another embodiment, the gHt is from herpes simplex
virus type 1 and comprises amino acid residues selected from the
group consisting of 1-792, 1-648, 1-475, 1-324 and 1-275.
[0013] In a further embodiment, the gHt is herpes simplex virus
type 1 gHt comprising amino acids 1-324.
[0014] In another embodiment, the gHt is herpes simplex virus type
1 gHt comprising amino acids 1-792 and the gL is herpes simplex
virus type 1 gL comprising amino acids 1-168.
[0015] The vaccine of the invention may further include a
substantially pure preparation of at least one of a herpes simplex
virus gD, gB or gC.
[0016] Also included in the invention is a vaccine comprising an
isolated DNA encoding a soluble herpes simplex virus gHt-gL complex
suspended in a pharmaceutically acceptable carrier.
[0017] In one embodiment of this aspect of the invention, the
vaccine includes an isolated DNA encoding at least one of a herpes
simplex virus gD, gB or gC.
[0018] The invention also relates to a herpes simplex virus
neutralizing antibody which specifically binds to a soluble herpes
simplex virus gHt-gL complex.
[0019] The invention further relates to an isolated DNA encoding a
herpes simplex virus neutralizing antibody which specifically binds
to a soluble herpes simplex virus gHt-gL complex.
[0020] There is also included in the invention a method of
immunizing a human patient against a herpes simplex virus infection
comprising administering to the patient a vaccine comprising a
soluble herpes simplex virus gHt-gL complex suspended in a
pharmaceutically acceptable carrier.
[0021] The invention further relates to a method of immunizing a
human patient against a herpes simplex virus infection comprising
administering to the patient a vaccine comprising an isolated DNA
encoding a soluble herpes simplex virus gHt-gL complex suspended in
a pharmaceutically acceptable carrier.
[0022] Also included in the invention is a method of treating a
herpes simplex virus infection in a human patient comprising
administering to the patient a vaccine comprising a soluble herpes
simplex virus gHt-gL complex suspended in a pharmaceutically
acceptable carrier.
[0023] The invention further includes a method of treating a herpes
simplex virus infection in a human patient comprising administering
to the patient a vaccine comprising an isolated DNA encoding a
soluble herpes simplex virus gHt-gL complex suspended in a
pharmaceutically acceptable carrier.
[0024] In each of the aforementioned methods of immunizing a human
patient or of treating a herpes simplex virus infection in a human
patient, the methods should be construed to optionally include the
administration of a substantially pure preparation of at least one
of a herpes simplex virus gD, gB or gC, or the administration of an
isolated DNA encoding at least one of a herpes simplex virus gD, gB
or gC.
[0025] There is further provided a method of treating a herpes
simplex virus infection in a human patient comprising administering
to the patient a herpes simplex virus neutralizing antibody which
specifically binds to a soluble herpes simplex virus gHt-gL complex
wherein the antibody is suspended in a pharmaceutically acceptable
carrier.
[0026] Also included is a method of treating a herpes simplex virus
infection in a human patient comprising administering to the
patient an isolated DNA encoding a herpes simplex virus
neutralizing antibody which specifically binds to a soluble herpes
simplex virus gHt-gL complex, wherein the DNA is suspended in a
pharmaceutically acceptable carrier.
[0027] In addition, the invention relates to a preparation of a
soluble herpes simplex virus gHt-gL complex and a substantially
pure preparation of a soluble herpes simplex virus gHt-gL
complex.
[0028] In a preferred embodiment, the herpes simplex virus is
selected from the group consisting of herpes simplex virus type 1
and herpes simplex virus type 2. Preferably, the herpes simplex
virus is herpes simplex virus type 1.
[0029] In one aspect, the complex is suspended in a
pharmaceutically acceptable carrier.
[0030] In another aspect, the gHt comprises amino acid residues
selected from the group consisting of 1-792, 1-648, 1-475, 1-324
and 1-275.
[0031] In yet another aspect, the gHt comprises amino acids
1-324.
[0032] In another aspect, the gHt comprises amino acids 1-792 and
the gL comprises amino acids 1-168.
[0033] The invention further relates to a cell, the DNA of the cell
encoding a soluble herpes simplex virus gHt-gL complex.
[0034] In one aspect, the cell is HL-7.
[0035] There is also provided in the invention a method of
modifying a cell to render it capable of secreting a soluble herpes
simplex virus gHt-gL complex comprising introducing into the cell
DNA encoding a truncated form of herpes simplex virus gH being gHt
and full length herpes simplex virus gL, wherein the gHt and the gL
are expressed in and secreted from the cell.
[0036] In one embodiment, the gHt comprises herpes simplex virus
type 1 gH comprising amino acid residues selected from the group
consisting of 1-792, 1-648, 1-475, 1-324 and 1-275.
[0037] The invention further relates to an isolated DNA comprising
DNA encoding a herpes simplex virus gHt and a substantially full
length herpes simplex virus gL.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a photograph of a gel depicting analysis of
carbohydrate processing in gH. Cytoplasmic extracts were prepared
from HSV-1 infected L cells, or from L cells transfected with
pCMV3gH-1 plus pCMV3gL-1 or with pCMV3gH-1 alone. Extracts were
subsequently treated with either endo H, endo F, or were untreated
and the proteins contained therein were resolved by SDS-PAGE,
transferred to a nylon membrane and incubated in the presence of
rabbit anti-gH serum (R83) and goat anti-rabbit horseradish
peroxidase conjugate. Reacted proteins were detected using a
chemiluminescent substrate. Abbreviations: gH, pCMV3gH-1; gL,
pCMV3gL-1; no tx, not treated.
[0039] FIG. 2 comprising panels A and B are photomicrographs
depicting analysis of folding of gH. L cells transfected with
pCMV3gH-1 (FIG. 2A) or cotransfected with pCMV3gH-1 and pCMV3gL-1
(FIG. 2B) were processed for immunofluorescence and were incubated
in the presence of the gH monoclonal antibody (MAb) 53S (American
Type Culture Collection), which antibody reacts with correctly
folded gH but does not react with incorrectly folded gH.
[0040] FIG. 3 is a series of photomicrographs depicting
intracellular localization of gH. L cells transfected with
pCMV3gH-1 (FIG. 3A) or cotransfected with pCMV3gH-1 and pCMV3gL-1
(FIG. 3B)) were processed for immunofluorescence and were reacted
with the gH MAb 37S, an antibody capable of reacting with gH
irrespective of its conformation.
[0041] FIG. 4 is a series of photomicrographs depicting analysis of
cell surface expression of gH and gL. L cells cotransfected with
pCMV3gH-1 and pMMTVgL-1 were incubated in the presence (Dex+) or
absence (Dex-) of 1 .mu.M dexamethasone (FIGS. 4A and 4B, or FIGS.
4C and 4D, respectively). In FIGS. 4E and 4F, cells were
cotransfected with pCMV3gH-1 and pCMV3gL-1. Expression of gH was
detected using the gH MAb 37S and anti-mouse IgG
fluorescein-labeled conjugate. Expression of gL was detected using
rabbit anti-gL serum and anti-rabbit IgG rhodamine-labeled
conjugate.
[0042] FIG. 5 is a series of photomicrographs depicting cellular
localization of gL. L cells were transfected as follows: pCMV3gL-1
alone (FIGS. 5A and 5B); pCMV3gL-1 plus pCMV3gH(792) (FIGS. 5C and
5D); or, pCMV3gL-1 plus pCMV3gH-1 (FIGS. 5E and 5F). Expression of
gL was detected using anti-gL serum and fluorescein-labeled
conjugate. Cells in the panels on the left were fixed with acetone
to examine internal localization of gL. Cells in the panels on the
right were not fixed in order to examine cell surface expression of
gL.
[0043] FIG. 6 is a photograph of a gel containing
immunoprecipitated gL to determine whether gL is secreted from
cells. L cells were transfected with the following plasmids:
pCMV3gL-1 alone; pCMV3gL-1 plus pCMV3gH(792); or, pCMV3gL-1 plus
pCMV3gH-1. Transfected cells were incubated in the presence of
.sup.35S-labeled cysteine. Supernatants were collected from cells
so incubated, which supernatants were concentrated and
immunoprecipitated with anti-gL serum. Immunoprecipitated proteins
were resolved by SDS-PAGE. Abbreviations: gL, pCMV3gL-1; gH(792),
pCMV3gH(792); gH, pCMV3gH-1.
[0044] FIG. 7 is a photograph of a gel depicting analysis of cell
supernatants for the presence or absence of secreted gL and
gH(792). L cells were transfected with pCMV3gL-1 plus pCMV3gH(792),
or with pCMV3gH(792) alone. Transfected cells were incubated in the
presence of .sup.35S-labeled cysteine. Supernatants were collected
from cells so incubated, which supernatants were concentrated and
immunoprecipitated with either gH MAb R83 or with anti-gL serum.
Immunoprecipitated proteins were resolved by SDS-PAGE.
Abbreviations: L, pCMV3gL-1; H, pCMV3gH(792).
[0045] FIG. 8 is a photograph of a gel depicting analysis of
carbohydrate processing of gL secreted by cells. L cells were
transfected with pCMV3gL-1 alone or with pCMV3gL-1 plus
pCMV3gH(792). Transfected cells were incubated in the presence of
.sup.35S-labeled cysteine. Supernatants were collected from cells
so incubated, which supernatants were concentrated and
immunoprecipitated with anti-gL serum. Immunoprecipitated proteins
were treated with endo H, endo F, or were untreated and the
products were resolved by SDS-PAGE. Abbreviations: L, pCMV3gL-1; H,
pCMVgH(792); no tx, no treatment.
[0046] FIG. 9 is a photograph of a gel depicting analysis of
complex formation between gL and various truncated mutant forms of
gH. L cells were cotransfected with pCMV3gL-1 and with one of the
following plasmids encoding truncated forms of gH as indicated at
the top of the gel: pCMV3gH(792); pCMV3gH(648); pCMV3gH(475); and,
pCMV3gH(102). Transfected cells were incubated in the presence of
.sup.35S-labeled cysteine. Supernatants were collected from cells
so incubated, which supernatants were concentrated and
immunoprecipitated with anti-gH serum. Immunoprecipitated proteins
were resolved by SDS-PAGE.
[0047] FIG. 10 is a diagram of the plasmids used to construct the
HL-7 cell line and diagrammatic representations of gHt and gL. HL-7
cells were obtained by co-transfecting mouse L cells with
pCMV3gH(792)-1, pCMV3gL-1 and pX343, which confers resistance to
hygromycin B (Blochlinger et al., 1984, Molecular & Cellular
Biology. 4:2929-2931). HL-7 was one of four separate clones which
expressed and secreted gHt-gL as a complex. The stick diagrams
illustrate major structural features of full length gH-1 and gL-1.
An arrow indicates the location of the truncation of gH at amino
acid 792. Balloons indicate positions of predicted N-linked
oligosaccharides and C indicates positions of cysteine residues.
The predicted signal peptide and transmembrane anchor regions are
indicated with shaded boxes.
[0048] FIG. 11 is a photograph of a gel depicting
immunoprecipitation of gH-gL complex secreted by HL-7 cells.
Proteins produced by HL-7 cells were labeled with
.sup.35S-cysteine, cell supernatants were obtained and were
immunoprecipitated with gH MAbs 52S, 53S and LP1 1 as indicated at
the top of the figure. As a control, proteins produced by L cells,
also labeled with .sup.35S-cysteine, were immunoprecipitated with
53S. Immunoprecipitated proteins were resolved by SDS-PAGE.
[0049] FIG. 12 is a photograph of an SDS-PAGE depicting
immunoaffinity purification of gHt-gL complex secreted from HL-7
cells. Lanes 1-3, 50 nanograms, 100 nanograms and 200 nanograms of
bovine serum albumin, respectively; lanes 4 and 5, 0.2 .mu.l and
1.0 .mu.l of pooled and concentrated 53S immunoaffinity column
purified gHt-gL complex, respectively.
[0050] FIG. 13 is a series of images of gels depicting
extracellular expression and purification of gHt-gL complex from
HL-7 cells. Lane 1 of each of FIGS. 13A, 13B and 13C contained 20
.mu.l of HL-7 cell supernatant. Lane 2 of each of FIGS. 13A, 13B
and 13C contained 20 .mu.l of flow through, and Lane 3 of each of
FIGS. 13A, 13B and 13C contained 1 .mu.g of protein eluted from the
53S immunoadsorbant column. In FIG. 13A, the samples were analyzed
by electrophoresis on a 10% SDS-polyacrylamide gel. The gel was
stained for protein with silver stain. In FIG. 13B, the proteins
were electrophoresed on a 10% SDS-polyacrylamide gel, transferred
to nitrocellulose and probed with anti-gL ascites 8H4. In FIG. 13C,
the proteins were electrophoresed on a 10% SDS-polyacrylamide gel,
transferred to nitrocellulose and probed with anti-gH serum
R83.
[0051] FIG. 14 depicts reactivity of purified gHt-gL with gH
specific antibodies. FIG. 14A is an image of a gel depicting
purified gHt-gL which was immunoprecipitated with either MAb LP11
(lane 1) or with .alpha.UL1-2 (lane 2) and was then electrophoresed
on a 10% SDS-polyacrylamide gel. The proteins were transferred to
nitrocellulose and were probed with R83 (anti-gH serum) (lane 1) or
with MAb 8H4 (anti-gL). FIG. 14B is a graph depicting reactivity of
gHt-gL with selected antibodies. Various concentrations of gHt-gL
were coated onto an ELISA plate for 2 hours at room temperature.
Wells were reacted with anti-gH MAbs LP11, 53S or 37S. Binding of
these antibodies was detected with horseradish peroxidase-labeled
goat anti-mouse antibody and ABTS substrate.
[0052] FIG. 15 is a series of graphs depicting the effect of gHt-gL
on HSV cell entry. Various concentrations of purified proteins
gC1(457t) (gCt), gD-1(306t) (gDt) or gHt-gL were added to Vero cell
monolayers in 96-well plates for 90 minutes at 4.degree. C. HSV-1
strain hrR3 was added at an MOI of 0.5 and the plate was incubated
for another 90 minutes at 4.degree. C. Plates were then shifted to
37.degree. C. for 5 hours. Cells were lysed and
.beta.-galactosidase activity was measured in aliquots of the
cytoplasmic extract using the substrate CPRG and measuring the
increase in absorption at 570 nm. In FIG. 15A, there is shown
blocking of virus entry with purified gCt, gDt or gHt-gL. In FIG.
15B, there is shown blocking of virus entry with gDt alone, gHt-gL
or a mixture of 40 nM gD (concentration which resulted in 50%
inhibition of virus entry) with various concentrations of
gHt-gL.
[0053] FIG. 16 is an image of two gels depicting immunoblot
(Western blot) analysis of serum samples from rabbits immunized
with gHt-gL. FIG. 16A: Purified gHt gL was electrophoresed on a
denaturing 10% SDS-polyacrylamide gel, transferred to
nitrocellulose and reacted with R136 (lane 1), R137 (lane 2), R138
(lane 3) or R139 (lane 4). FIG. 16B: Purified gHt-gL was
electrophoresed on a non-denaturing (native) 10% SDS-polyacrylamide
gel, transferred to nitrocellulose and reacted with R136 (lane 1),
R137 (lane 2), R138 (lane 3) or R139 (lane 4).
[0054] FIG. 17 is a series of graphs depicting blocking of HSV
entry into cells by rabbit antibodies specific for gHt-gL. FIG.
17A: HSV-1 hrR3 was incubated for 90 minutes at 37.degree. C. with
various concentrations of rabbit anti-gHt-gL sera R136, R137, R138,
or R139. The serum-virus mixture was added to Vero cell monolayers
in a 96 well plate, incubated at 4.degree. C. for 90 minutes
followed by incubation at 37.degree. C. for 5 hours. Virus entry
was assayed as an increase in .beta.-galactosidase activity in
cytoplasmic extracts obtained from each well and expressed as % of
control values obtained with virus alone. FIG. 17B: HSV-1 hrR3 was
added to Vero cell monolayers at 4.degree. C. for 90 minutes. The
medium was removed and various dilutions of either R83, R137 or
LP11 were added. The monolayers were incubated at 4.degree. C. for
90 minutes followed by incubation at 37.degree. C. for 5 hours.
Virus entry was assayed as described in FIG. 17A.
[0055] FIG. 18 is an image of immunoblot (western blot) analysis of
cytoplasmic extracts of HSV-1 or HSV-2 infected Vero cells. Samples
of purified gHt-gL or cytoplasmic extracts were electrophoresed on
a denaturing 10% SDS-polyacrylamide gel, transferred to
nitrocellulose and reacted with R137 (lanes 1-4) or mouse
anti-gHt-gL (lanes 5-8). The mouse serum was pooled from 9 animals
immunized with gHt-gL Experiment I, Table 2). gHt-gL purified from
HL-7 cells (lanes 1 and 4) were included on the gel as a control.
Cytoplasmic extracts were prepared from uninfected cells (lanes 2
and 6) or from cells infected with HSV-1 (NS) (lanes 3 and 7) or
HSV-2 (333) (lanes 4 and 8).
[0056] FIG. 19 is a series of graphs depicting blocking of HSV
entry by mouse antibodies to gD or to gHt-gL. FIG. 19A: HSV-1 hrR3
was incubated for 90 minutes at 37.degree. C. with various
concentrations of antisera obtained from mice immunized either with
full-length gD (obtained from HSV-1 infected cells) or with gHt-gL
(obtained from HL-7 cells) or with PBS according to Experiment I
(Table 2). The serum-virus mixture was added to Vero cell
monolayers in a 96 well plate, incubated at 4.degree. C. for 90
minutes and then at 37.degree. C. for 5 hours. Virus entry was
assayed as an increase in .beta.-galactosidase activity in
cytoplasmic extracts from each well and expressed as % of control
values obtained with virus alone. FIG. 19B: This is identical to
FIG. 19A except that the sera were obtained from mice immunized as
part of Experiment II (Table 2). Each of the sera from both
experiments were assayed and only one representative curve for each
experimental group is shown. All of the sera in each group yielded
similar curves.
DETAILED DESCRIPTION OF THE INVENTION
[0057] The invention relates to the discovery that two HSV-1
specific glycoproteins, gH and gL, when complexed together and
administered to animal, serve to protect the animal against
infection by HSV. Thus, there has been discovered a subunit vaccine
comprising a soluble HSV-1 gHt-gL complex, which vaccine is useful
not only as a prophylactic therapeutic agent for protection of an
animal against a herpesvirus infection, but is also useful as a
therapeutic agent for treatment of an ongoing herpesvirus infection
in an animal, particularly in an animal having a high propensity to
reactivate a herpesvirus infection.
[0058] It is known that HSV-1 gH and gL form a molecular complex
which is present on the virion envelope. This complex is essential
for viral infectivity in that it is required for entry of virus
into cells and for cell to cell spread of virus which is believed
to occur via membrane fusion. In the experiments described herein,
gH and gL have been stably expressed in and secreted from mammalian
cells in culture as a soluble complex, named gHt-gL. This complex,
when inoculated into an animal, elicits antibody which serves to
neutralize virus in a virus neutralization cell culture assay.
Further, when the gHt-gL complex is inoculated into an animal, it
elicits an immune response which serves to protect the inoculated
animal against disease when the animal is challenged with
infectious virus. Thus, it has been discovered according to the
present invention that a soluble herpes simplex virus type 1 gHt-gL
complex functions to vaccinate an animal against herpes simplex
virus disease.
[0059] By the term "soluble gHt-gL complex" as used herein, is
meant a complex comprising a truncated HSV gH and a substantially
full length HSV gL which are bound together in the complex and
which are soluble in an aqueous solution.
[0060] The soluble gHt-gL complex of the invention may be obtained
in large quantities for use as a vaccine for protection of humans
against HSV infection, or for eliminating or diminishing the
frequency of reactivation of the virus from the latent state thus,
reducing the severity of recurrent HSV infection in humans. The
complex is also useful as a diagnostic reagent for assessing the
presence or absence of a herpesvirus infection in a human. Such an
assessment is made by obtaining serum from the individual and
reacting it with the complex in a standard immunoassay such as
radioimmunoassay or enzyme linked immunoadsorbent assay
(ELISA).
[0061] By the term "vaccine" as used herein, is meant a
composition, a protein complex or a DNA encoding a protein complex
which serves to protect an animal against a herpesvirus
disease.
[0062] By the term "immunizing a human against herpes simplex virus
infection" is meant administering to the human a composition, a
protein complex, a DNA encoding a protein complex, an antibody or a
DNA encoding an antibody, which elicits an immune response in the
human which immune response provides protection to the human
against a herpes simplex virus disease.
[0063] Homologs of the genes encoding HSV-1 gH and gL have been
identified in most other herpesviruses including human CMV (Cranage
et al., 1988, J. Virol. 62:1416), VZV (Davison and Scott, 1986, J.
Gen. Virol. 67:1759) and EBV (McGeoch and Davison, 1986, Nucl.
Acids Res. 4:4281). The CMV UL115 gene, a positional homolog of the
HSV-1 gL gene, encodes a secreted protein which forms a complex
with CMV gH and is therefore a positional and likely functional
(although not a sequence) homolog to HSV-1 gL (Kaye et al., 1992,
J. Gen. Virol. 73:2693; Spaete et al., 1993, Virology 193:853).
HHV-6 (Josephs et al., 1991, J. Virol. 65:5597), pseudorabies virus
(Klupp et al., 1991, Virology 182:732) and herpesvirus saimiri
(Gompels et al., 1988, J. Gen. Virol. 69:2819) also each encode
homologs of HSV-1 gH and gL.
[0064] The invention should not be construed to be limited to a
soluble HSV-1 gHt-gL complex. Rather, the invention should be
construed to encompass soluble gHt-gL complexes which are derived
from both HSV-1 and HSV-2, which soluble complexes may be used as
vaccines to protect humans from disease caused by either of these
two types of viruses. As the data presented herein establish,
antibody directed against soluble HSV-1 gHt-gL complex serves to
neutralize infection of cells in culture by HSV-2. Thus, since
antibodies raised against HSV-1 gHt-gL complex neutralize HSV-2,
the invention should be construed to include gHt-gL complexes from
either virus type which serve to protect cells and humans against
infection by both HSV-1 and HSV-2.
[0065] The gHt-gL complex of the invention may therefore comprise
one subunit derived from HSV-1 and another subunit derived from
HSV-2, yielding at least four general classes of complexes which
are encompassed by the invention. One complex comprises HSV-1 gH
bound to HSV-1 gL. Another complex comprises HSV-1 gH bound to
HSV-2 gL. A third complex comprises HSV-2 gH bound to HSV-1 gL and
a fourth complex comprises HSV-2 gH bound to HSV-2 gL.
[0066] The gHt-gL complex of the invention comprises a truncated gH
molecule which is complexed to a substantially full length gL
molecule. It has been discovered in the present invention that it
is necessary that the gH portion of the gHt-gL complex be truncated
in order that the complex is secreted from the cell in soluble
form. Truncated forms of gH (referred to herein as "gHt") include
those containing amino acid residues selected from the group
consisting of 1-792, 1-648, 1-475, 1-324 and 1-275.
[0067] As is customary in the field of herpes simplex virology,
amino acids in proteins encoded by herpes simplex viruses are
numbered from the first methionine in the protein.
[0068] By the term "truncated" as used herein as it refers to gH,
is meant a molecule of gH which contains less than the complete
number of amino acids found in a wild type protein. Particularly,
the term truncated is used to mean a gH molecule which is not
membrane anchored, i.e., which comprises a deletion or other
mutation which facilitates secretion of gH from the cell. Mutations
in the gH molecule which give rise to different lengths of gH may
comprise insertion, deletion or point mutations. An insertion
mutation is one where additional base pairs are inserted into a DNA
molecule. A deletion mutation is one where base pairs have been
removed from a DNA molecule. A point mutation is one where a single
base pair alteration has been made in a DNA molecule. Each of these
mutations is designed such that creation of any one of them in a
DNA molecule effects an alteration in the nature of any polypeptide
expressed by that DNA, which alteration results in a gH molecule
capable of binding to gL to form a complex having biological
activity as defined herein, and which gH-gL complex is secreted
from a cell in which it is expressed.
[0069] The complex also includes a substantially full length gL
molecule which may comprise all of the amino acids of gL, or may
also be mutated comprise less than all of the amino acids of
gL.
[0070] By the term "substantially full length herpesvirus gL" as
used herein, is meant a herpesvirus gL molecule which comprises a
sufficient number of amino acids so that the substantially full
length gL is capable of binding to gHt, forming a complex
therewith, which complex has biological activity as defined herein.
Thus, a substantially full length gL molecule does not necessarily
contain all of the amino acids which comprise herpesvirus gL,
(although according to the invention, it may) but rather, the
molecule comprises a substantial portion of the molecule sufficient
for binding to gHt and forming a biologically active complex
therewith.
[0071] Referring to gH and gL molecules encoded by HSV-1, it has
been discovered in the present invention that a stable gHt-gL
complex can be formed wherein the gHt component comprises amino
acids 1-324 and the gL component is substantially full length. A
stable gHt-gL complex can also be formed wherein the gHt component
comprises amino acids amino acids 1-792 and the gL component
comprises amino acids 1-168. Thus, the invention should not be
construed to be limited to any particular specific length of either
gH or gL. Rather, the invention should be construed to encompass
any length of a truncated gH which binds to any length of gL to
form a complex which has gHt-gL biological activity as defined
herein. The procedures which are used to generate plasmids
expressing proteins of different lengths are well known in the art
and the means for expressing gH and gL in a cell such that they
form a biologically active complex are described in detail herein.
Thus, it is well within the skill of those in the art to generate
biologically active gHt-gL complexes, wherein the gHt-and gL each
comprise an animo acid length which is different from the gHt and
gL molecules disclosed in the experimental examples section
herein.
[0072] The invention should also be construed to include any form
of a gHt-gL complex having substantial homology to the HSV-1 gHt-gL
complex disclosed herein. Preferably, a gHt-gL complex which is
substantially homologous is about 50% homologous, more preferably
about 70% homologous, even more preferably about 80% homologous and
most preferably about 90% homologous to the gHt-gL complex secreted
from HL-7 cells.
[0073] "Homologous" as used herein, refers to the subunit sequence
similarity between two polymeric molecules, e.g., between two
nucleic acid molecules, e.g., two DNA molecules or two RNA
molecules, or between two polypeptide molecules. When a subunit
position in both of the two molecules is occupied by the same
monomeric subunit, e.g., if a position in each of two DNA molecules
is occupied by adenine, then they are homologous at that position.
The homology between two sequences is a direct function of the
number of matching or homologous positions, e.g., if half (e.g.,
five positions in a polymer ten subunits in length) of the
positions in two compound sequences are homologous then the two
sequences are 50% homologous, if 90% of the positions, e.g., 9 of
10, are matched or homologous, the two sequences share 90%
homology. By way of example, the DNA sequences 3'ATTGCC 5' and
3'TAAGCC 5' share 50% homology. Also by way of example, the amino
acid sequences CTAGYR and CTACRY share 50% homology.
[0074] To generate an HSV gHt-gL complex, following the teaching
provided herein it is well within the skill of those in the art to
take a plasmid encoding truncated gH and full length gL from other
strains of HSV-1 and introduce it into a population of mammalian
cells such that the cells become stably transfected with the
plasmid and are caused to express and secrete a soluble form of
gHt-gL complex as described herein. It is also well within the
skill of those in the art to take yet another plasmid encoding gH
and gL (i.e., DNA obtained from various strains of HSV-2, which DNA
encodes gH and gL) and generate cell lines which secrete soluble
gHt-gL complex following the teaching contained herein.
[0075] The invention should not be construed to be limited to the
particular method of introduction of herpesvirus DNA into mammalian
cells described herein. Rather, other methods may be used to
generate cells which express a soluble form of gH-gL complex. Such
methods include, but are not limited to, the use of retroviral and
other viral vectors for delivery of herpesvirus-specific gH-gL
encoding DNA into cells and the use of other chemical means of
transfection. In addition as described herein, the complex to be
formed by cells encoding gH-gL may include a mixture of gH derived
from one virus strain and gL derived from yet another virus strain.
Generation of such mixed complexes is accomplished using the
protocols described above and other protocols available to
virologists, described for example in Sambrook et al. (1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y.).
[0076] The invention should also be construed to include gHt-gL
complexes which are generated in mammalian cells as described
herein, or which are generated by other means, such as by
expression in a baculovirus system, or a yeast expression system.
gHt-gL complexes which are generated by synthetic methods are also
included in the invention.
[0077] Also contemplated by the invention is a subunit vaccine
comprising an isolated nucleic acid, preferably, an isolated DNA,
encoding a gHt-gL complex. Such a nucleic acid, preferably, a DNA
molecule, may be used directly as a vaccine as described herein, or
it may be used to transfect cells in order to produce large
quantities of gHt-gL for use as a subunit vaccine.
[0078] To generate a DNA encoding gHt-gL, the desired gHt and gL
coding sequences are ligated together in either of two
configurations. In the first configuration, a plasmid is generated
having the following elements: a promoter for expression of gHt
which is positioned upstream of a desired gHt encoding sequence and
a promoter for expression of gL which is positioned upstream of a
desired gL coding sequence. The plasmid therefore encodes gHt and
gL on the same molecule wherein expression of each of gHt and gL is
under the control of an individual promoter sequence, preferably
the same promoter sequence. Both gHt and gL are expressed
individually from this plasmid in a cell and form complex therein
which is secreted from the cells as described herein.
[0079] Alternatively, a plasmid may be generated which has the
following elements: a single a promoter which is positioned
upstream of a desired gHt encoding and a desired gL encoding
sequence, the gHt and gL encoding sequences being separated by a
DNA sequence encoding a cleavage site for a protease. In this
plasmid, the gHt and gL encoding sequences may be positioned in the
plasmid in either orientation which respect to each other, such
that either one of them is juxtaposed to the promoter sequence. DNA
encoding the protease cleavage site which is positioned between the
gHt and gL coding sequences may be any DNA known to encode a length
of amino acids which are cleaved by any protease which is present
in a majority of cells and which is particularly present in cells
into which the DNA of the invention is introduced. gHt-gL which is
expressed by this plasmid is initially expressed in a cell as a
single length of protein comprising gHt and gL fused together via a
protease cleavage site. Subsequent cleavage of the fused protein by
a protease generates individual molecules of gHt and gL which form
a complex which is secreted from the cell as desccribed herein.
[0080] The isolated DNA of the invention is not limited to a
plasmid based DNA, but rather may include any form of DNA which
encodes gHt-gL as described herein in the case of a plasmid DNA.
Thus, the isolated DNA of the invention may include a viral vector,
a non-viral vector, or a plasmid DNA.
[0081] The promoter sequence which is used to drive expression of
gHt-gL in either type of configuration may be any constitutive
promoter which drives expression of these proteins in cells. Such
promoters therefore include, but are not limited to, the
cytomegalovirus immediate early promoter/regulatory sequence, the
SV40 early promoter/enhancer sequence, the Rous sarcoma virus
promoter/enhancer and any other suitable promoter which is
available in the art for constitutive expression of high levels of
proteins in cells.
[0082] When the isolated DNA of the invention is used to generate
large quantities of gHt-gL complex, cells are transfected with the
DNA using the methodology disclosed herein or any other available
transfection methodology, gHt-gL is expressed and is recovered from
the cells as described herein.
[0083] When the isolated DNA is to be used as a vaccine, a DNA
based vaccine is prepared following the disclosure described in
Wang et al. (1993, Proc. Natl. Acad. Sci. USA 90:4156-4160). The
vaccine comprises DNA encoding a gHt and a substantially full
length gL expressed under the control of any of the promoters
disclosed herein. Antibodies are raised against the expressed
protein by intramuscular injection of DNA into the hind limb of six
to eight week old mice. The anesthetic bupivacaine (50 .mu.l of a
0.5% solution) is used to improve immunogenicity of the vaccine.
The animals are immunized first with bupivacaine and then are
immunized the following day with 50 .mu.g of plasmid DNA encoding
gHt-gL. At about four weeks, animals are test bled to measure the
level of anti-gHt-gL antibody and are re-injected with bupivacaine
and DNA on successive days. On day 45, or thereabouts, serum is
collected from the animals and is tested to determine whether
antibodies contained therein neutralize virus in the virus
neutralization assays described herein. DNA encoding other HSV
glycoproteins such as, but not limited to gD, gC and gB may
included for immunization of the animal using the same
protocol.
[0084] To adapt this DNA based vaccine to human subjects, the
amounts of DNA, the route of injection and the adjuvants to be used
may vary from that just described. However, these variations will
be readily apparent to the skilled artisan working in the field of
DNA based vaccines.
[0085] The invention should be construed to include any and all
isolated DNAs which are homologous to the gHt-gL DNA described and
referenced herein, provided these homologous DNAs have the
biological activity of gHt-gL complex as defined herein.
[0086] An "isolated DNA", as used herein, refers to a DNA sequence,
segment, or fragment which has been purified from the sequences
which flank it in a naturally occurring state, e.g., a DNA fragment
which has been removed from the sequences which are normally
adjacent to the fragment, e.g., the sequences adjacent to the
fragment in a genome in which it naturally occurs. The term also
applies to DNA which has been substantially purified from other
components which naturally accompany the DNA, e.g., RNA or DNA or
proteins which naturally accompany it in the cell.
[0087] The invention should also be construed to include DNAs which
are substantially homologous to the DNA isolated according to the
method of the invention. Preferably, DNA which is substantially
homologous is about 50% homologous, more preferably about 70%
homologous, even more preferably about 80% homologous and most
preferably about 90% homologous to DNA obtained using the method of
the invention.
[0088] The invention should therefore be construed to include any
form of a gHt-gL complex or DNA encoding a gHt-gL complex, which is
homologous to the HSV-1 gHt-gL complex or it's DNA disclosed herein
and which has or encodes gHt-gL complex biological activity as
defined herein.
[0089] To purify a gHt-gL complex for use as a vaccine or other
therapeutic, the examples given in the experimental details section
may be followed. Essentially, a substantially pure preparation of a
gHt-gL complex is obtained by immunoaffinity chromatography of
supernatants obtained from cells which express and secrete gHt-gL
complex using the monoclonal antibody (MAb), 53S or any other
antibody which specifically binds gH, gL or the combination of the
two. To purify the gH-gL complex, the supernatant is passed over an
affinity column comprising anti-gHt-gL complex antibody, the column
is washed with buffer and adsorbed proteins are eluted from the
column in fractions using an elution buffer, such as 50 mM glycine
buffer (pH 2.5) containing 0.5 M NaCl and 0.1% Triton X-100.
Fractions so eluted are neutralized with a high pH buffer, for
example, Tris-HCl, pH 9.0 and are then analyzed for the presence of
gHt and gL by gel electrophoresis or other protein detection
technology. Fractions containing the proteins are pooled and are
concentrated using a commercially available concentrator, for
example, a Centricon-10 concentrator.
[0090] By the term "substantially pure" as it refers to a gH-gL
complex, is meant a complex which has been separated from the
components which naturally accompany it in the cell or medium in
which it resides. Typically, a compound is substantially pure when
at least 10%, more preferably at least 20%, more preferably at
least 50%, more preferably at least 60%, more preferably at least
75%, more preferably at least 90%, and most preferably at least 99%
of the total material (by volume, by wet or dry weight, or by mole
percent or mole fraction) in a sample is the compound of interest.
Purity can be measured by any appropriate method, e.g., in the case
of polypeptides by column chromatography, gel electrophoresis or
HPLC analysis. A compound, e.g., a protein, is also substantially
purified when it is essentially free of naturally associated
components or when it is separated from the native contaminants
which accompany it in its natural state.
[0091] The invention should be construed to include modifications
of gHt or gL which in their modified form are capable of forming a
complex having the biological activity of the gHt-gL complex
disclosed herein. For example, conservative amino acid
substitutions may be made in either or both of gHt or gL which
alter the primary sequence of the proteins without significantly
affecting the ability of these proteins to bind together and retain
the biological activity of the gHt-gL complex. Conservative amino
acid substitutions typically include substitutions within the
following groups, but are not limited to these groups: glycine,
alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine;
phenylalanine, tyrosine. Also included are proteins and peptides
which have been modified using ordinary molecular biological
techniques so as to improve their resistance to proteolytic
degradation, to optimize solubility properties, or to alter
post-translational modification of the protein or peptide. The
proteins and peptides of the invention are not limited to products
of any of the specific exemplary processes listed herein.
[0092] By "biological activity" of gHt-gL as used herein, is meant
a gHt-gL complex which when inoculated into an animal elicits an
antibody, a virus neutralizing antibody, which neutralizes the
infectivity of a herpesvirus in a virus neutralization assay and
which protects an animal against disease when wild type virus is
subsequently administered to the animal.
[0093] Typically, a virus neutralization assay involves incubation
with a known titer of infectious virus of serial dilutions of serum
obtained from an animal administered the vaccine for a period of
time. Following the incubation period, the amount of infectious
virus remaining is quantitated, usually by plaque assay.
[0094] The term "virus neutralizing effective amount" as used
herein, means an amount of antigen which elicits an immune response
when administered to an animal, which response is capable of
neutralizing virus infectivity to a level which is less than 50% of
normal infectivity in a standard virus neutralization assay.
[0095] A virus neutralizing immune response is also one which
affords protection to the animal from lethal challenge with wild
type virus. Protection against lethal challenge with wild type
virus is typically assessed by first immunizing a series of animals
with the subject antigen to generate serum capable of neutralizing
virus infectivity in a standard virus neutralization assay. The
animals are then inoculated with a serial dilutions of wild type
virus, which dilutions contain sufficient virus to kill
non-immunized animals. The death rate of the animals is quantitated
and is compared to the level of the virus neutralizing immune
response in each of the animals. Protection from lethal challenge
has been effected when non-immunized animals die and immunized
animals do not die as a result of infection with virus.
[0096] Also included in the term "virus neutralizing immune
response" is a response which affords protection against HSV
disease, such as zosteriform disease, the establishment of latency
in ganglia that innervate the site of infection and the production
of subclinical (asymptomatic) virus shedding. Protection against
HSV disease is typically assessed by counting HSV lesions that
develop at the site of virus challenge, or by scoring animals for
erythema (redness) and swelling. In the zosteriform model, disease
is scored as primary, i.e., at the site of infection, or secondary,
i.e., at a site remote from the initial site of infection but along
the axis of the same dermatome.
[0097] Protection against the establishment of latency is typically
assessed by removing the ganglia and determining the presence (and
amount) or absence of HSV in the ganglia. Protection against
subclinical virus shedding is typically determined by culturing
virus from the animal at selected intervals post-challenge or by
using standard PCR to measure levels of viral DNA.
[0098] By the term "virus neutralizing antibody" as used herein, is
meant a reduction in the infectivity of a virus in the presence of
the antibody compared with the infectivity of the virus in the
absence of the antibody. Typically, an antibody is a virus
neutralizing antibody when the infectivity of the virus is reduced
by about 50% in the presence of the antibody at a dilution of the
serum containing the antibody which is greater than 1:20. The
higher the dilution of serum which neutralizes a constant amount of
virus by 50%, the greater the estimate of the activity of the
antibody contained within the serum.
[0099] The term "protect an animal against disease" is used herein
to mean a reduction in the level of disease caused by a wild type
virus in an animal inoculated with a gHt-gL complex compared with
the level of disease caused by a wild type virus in an animal which
as not been inoculated with a gHt-gL complex. As the data presented
herein establish, the gHt-gL complex of the invention protected an
animal against infection by HSV to the same extent as did gD when
animals were similarly administered this glycoprotein. As noted
herein, HSV gD as a subunit vaccine is currently in clinical trials
and at present represents the "gold standard" of HSV vaccines.
Thus, the gHt-gL subunit vaccine of the invention is capable of
protecting an animal against HSV disease to a level at least as
good as that observed when gD is used to immunize an animal.
[0100] To determine whether a gHt-gL complex generated using the
methods described herein has biological activity, the following
general protocols are followed. To assess biological activity of a
gHt-gL complex, an animal is first immunized with the complex.
Although the examples provided herein are directed to rabbits and
mice, any other animal may be used.
[0101] Using mice as an example, a mouse is immunized at about
biweekly intervals with about four doses of approximately 10 .mu.g
of gHt-gL per dose. Serum obtained from the mouse post-immunization
is tested for the presence of an anti-gHt-gL complex antibody in
any immunological assay, for example, an ELISA. A virus
neutralization assay is performed wherein dilutions of serum
obtained from the immunized animal are mixed with infectious virus.
The mixture is added to cells and neutralization of virus by the
antibody is measured as described herein.
[0102] To determine the efficacy of the gH-gL complex as a vaccine,
the gH-gL complex is administered intraperitoneally to mice using
an adjuvant system suitable for administration of proteins to mice,
for example, the Ribi adjuvant system (RAS; Ribi Immunochemical
Research, Hamilton, Mont.), or other suitable adjuvant. Both pre-
and post-immune serum is obtained from the mice and the presence or
absence of antibodies is determined in the standard assays
described herein. The ability of anti-gHt-gL antibodies to
neutralize HSV is determined in a standard viral neutralization
assay, such as but not limited to, a plaque reduction
neutralization assay. Mice are administered a range of
concentrations of gH-gL complex from about 0.1 to about 20 .mu.g
per dose, using several different immunization schedules, i.e.,
weekly, biweekly, in order to determine the optimum conditions for
effective immunization of the mice against HSV. Sera obtained from
mice so immunized are tested for the ability to neutralize HSV-1
strain NS (or other strain of HSV depending on the virus from which
the gHt-gL complex is derived) and other strains of both HSV-1 and
HSV-2. Since the ability of an antibody to neutralize virus in
culture is predictive of the protective activity of that antibody,
neutralization of any one of the viruses listed above by antibody
raised against the gHt-gL complex is predictive of the ability of
gHt-gL complex to serve as a subunit vaccine candidate against that
virus.
[0103] To assess whether antibody raised against gH-gL protects
mice against in vivo challenge with virus, immunized and
non-immunized mice are administered various concentrations of virus
intraperitoneally at a time post-immunization when peak antibody
levels are apparent following the experiments described above. The
number of immunized animals which survive challenge by virus is
indicative of the efficacy of the gHt-gL complex as a vaccine
candidate. Although these studies may be conducted using an
intraperitoneal route, studies on the vaccine capabilities of a
gHt-gL complex may involve all possible routes of administration
including, but not limited to, intramuscular, subcutaneous and even
oral routes of administration. In addition, as described in the
experimental details section herein, other animal models for
herpesvirus infections, such as guinea pigs are used.
[0104] Furthermore, studies may be conducted to examine viral
latency in gH-gL immunized animals surviving virus challenge and in
animals which are administered the complex and are then tested in
any of the available latency models of HSV infection. Such studies
will be performed according to published protocols, such as that
described by Stanberry (Pathogenesis of herpes simplex virus
infection and animal models for its study. In: Current Topics in
Microbiology and Immunology, 179: Herpes simplex virus:
Pathogenesis and Control, Springer Verlag (Berlin), 1992, pp
15-30). Thus, the establishment of latency in ganglia that
innervate the site of infection and the production of subclinical
(asymptomatic) virus shedding may be examined as described
herein.
[0105] The subunit vaccine of the invention may be formulated to be
suspended in a pharmaceutically acceptable composition suitable for
use in animals and in particular, in humans. Such formulations
include the use of adjuvants such as muramyl dipeptide derivatives
(MDP) or analogs which are described in U.S. Pat. Nos. 4,082,735;
4,082,736; 4,101,536; 4,185,089; 4,235,771; and, 4,406,890. Other
adjuvants which are useful include alum (Pierce Chemical Co.),
lipid A, trehalose dimycolate and dimethyldioctadecylammonium
bromide (DDA), Freund's adjuvant, and IL-12. Other components may
include a polyoxypropylene-polyoxyethylene block polymer
(Pluronic.RTM.), a non-ionic surfactant, and a metabolizable oil
such as squalene (U.S. Pat. No. 4,606,918).
[0106] The subunit vaccine of the invention may be encapsulated
into liposomes for administration to the animal. See for example,
U.S. Pat. Nos. 4,053,585, 4,261,975 and 4,406,890.
[0107] The subunit vaccine of the invention is administered to a
human by any suitable route of administration, for example,
subcutaneously, intramuscularly, orally, intravenously,
intradermally, intranasally or intravaginally. The complex is first
suspended in a pharmaceutically acceptable carrier which is
suitable for the chosen route of administration and which will be
readily apparent to those skilled in the art of vaccine preparation
and administration. The dose of vaccine to be used may vary
dependent upon any number of factors including the age of the
individual and the route of administration. Typically, the subunit
vaccine is administered in a range of 1 .mu.g to 50 mg of protein
per dose. Approximately 1-10 doses are administered to the
individual at intervals ranging from once per day to once per week
to once every few years.
[0108] The vaccine of the invention is useful for prevention of
herpesvirus disease in an animal, preferably a human. However, the
vaccine is also useful as a therapeutic agent for treatment of
acute episodes of herpesvirus infection in order to boost the
immune response in the animal. Thus the invention contemplates both
prophylactic and therapeutic uses for the vaccine of the
invention.
[0109] It should be appreciated that the subunit vaccine of the
invention may be combined with other subunit vaccines, such as
subunit vaccines comprising gD, gB or combinations thereof, gC, and
the like each of which may be generated and used according to
published protocols and the procedures described herein.
[0110] The antibodies which are produced in animals may themselves
serve as therapeutic compounds for treatment of HSV infection,
particularly in severely immunocompromised individuals, such as
those infected with human immunodeficiency virus or those receiving
transplants. The antibody may also be useful for administration to
newborn infants infected with HSV and to adults at risk for
developing HSV encephalitis. The invention should therefore be
construed to include anti-gHt-gL antibodies as described herein and
anti-gHt-gL antibodies which may be modified such that they are
phage displayed and/or humanized using technology available in the
art. It will be appreciated that the antibodies which are useful
include those which specifically bind to a gHt-gL complex derived
from either HSV-1 or HSV-2 and a gHt-gL complex wherein one
component id derived from HSV-1 and the other component is derived
from HSV-2. Similarly, DNA encoding antibodies which are now
described may comprises DNAs encoding gHt and gL subunits derived
from either of HSV-1 or HSV-2.
[0111] The generation of polyclonal and monoclonal antibodies is
well known in the art and is described and referenced herein. Phage
displayed and humanized antibodies are also well known in the art
and are also described herein.
[0112] Given the advances in technology in cloning DNA encoding
proteins comprising antibodies, the invention should also be
construed to include an isolated DNA which encodes a gHt-gL
antibody, or a portion or fragment of such antibody.
[0113] When the antibody of the invention is a monoclonal antibody,
the nucleic acid encoding the antibody may be cloned and sequenced
using technology which is available in the art, and is described,
for example, in Wright et al. (1992, Critical Rev. in Immunol.
12(3,4):125-168) and the references cited therein. Further, the
antibody of the invention may be "humanized" using the technology
described in Wright et al., (supra) and in the references cited
therein.
[0114] For example, to generate a phage antibody library, a cDNA
library is first obtained from mRNA which is isolated from cells,
e.g., the hybridoma, which express the desired protein to be
expressed on the phage surface, e.g., the desired antibody. cDNA
copies of the mRNA are produced using reverse transcriptase. cDNA
which specifies immunoglobulin fragments are obtained by PCR and
the resulting DNA is cloned into a suitable bacteriophage vector to
generate a bacteriophage DNA library comprising DNA specifying
inmunoglobulin genes. The procedures for making a bacteriophage
library comprising heterologous DNA are well known in the art and
are described, for example, in Sambrook et al. (1989, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).
[0115] Bacteriophage which encode the desired antibody, may be
engineered such that the protein is displayed on the surface
thereof in such a manner that it is available for binding to its
corresponding binding protein, e.g., the antigen against which the
antibody is directed. Thus, when bacteriophage which express a
specific antibody are incubated in the presence of a cell which
expresses the corresponding antigen, the bacteriophage will bind to
the cell. Bacteriophage which do not express the antibody will not
bind to the cell. Such panning techniques are well known in the art
and are described for example, in Wright et al., (supra).
[0116] Processes such as those described above, have been developed
for the production of human antibodies using M13 bacteriophage
display (Burton et al., 1994, Adv. Immunol. 57:191-280).
Essentially, a cDNA library is generated from mRNA obtained from a
population of antibody-producing cells. The mRNA encodes rearranged
immunoglobulin genes and thus, the cDNA encodes the same. Amplified
cDNA is cloned into M13 expression vectors creating a library of
phage which express human Fab fragments on their surface. Phage
which display the antibody of interest are selected by antigen
binding and are propagated in bacteria to produce soluble human Fab
immunoglobulin. Thus, in contrast to conventional monoclonal
antibody synthesis, this procedure immortalizes DNA encoding human
immunoglobulin rather than cells which express human
immunoglobulin.
[0117] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage as
described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using synthetic DNA or amino acid sequence technology which is
available and well known in the art.
[0118] By the term "specifically binds," as used herein, is meant
an antibody which recognizes and binds an HSV gHt-gL complex, but
does not substantially recognize or bind other molecules in a
sample.
[0119] The invention thus includes an isolated DNA encoding a
gHt-gL antibody or a portion of the antibody of the invention. To
isolate DNA encoding an antibody, for example, DNA is extracted
from antibody expressing phage obtained according to the methods of
the invention. Such extraction techniques are well known in the art
and are described, for example, in Sambrook et al. (supra).
[0120] The anti-gHt-gL complex antibody of the invention may be
conventionally administered to a mammal, preferably a human,
parenterally, by injection, for example, subcutaneously,
intravenously, intramuscularly, and the like. Additional
formulations which are suitable for other modes of administration
include suppositories, intranasal aerosols and, in some cases, oral
formulations. The antibody may be administered in any of the
described formulations either daily, several times daily, weekly,
bi-weekly or monthly or several times a year in a dosage which will
be apparent to the skilled artisan and will depend on the type of
disease being treated. Preferably, the dosage will range from about
1 nanogram of antibody to several milligrams of antibody to even up
to about 100 milligrams of antibody per dose.
[0121] It will be appreciated that the subunit vaccine of the
invention, the DNA vaccine of the invention and the antibody of the
invention may be used to prevent or treat HSV infections in a human
in cases where the human is not yet infected, in cases where the
human is infected and treatment is initiated in order to prevent
more severe infection, such as, for example, HSV encephalitis, and
in cases where the human is latently infected with the virus and
has a high propensity to reactivate. In addition, the compositions
of the invention are useful for treatment of neonates at risk for
developing severe herpesvirus infection and immunosuppressed
individuals at risk for developing severe herpes virus infection,
such as is the case in patients having acquired immunodeficiency
syndrome and in transplant patients and those requiring
chemotherapy.
[0122] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
EXAMPLE 1
[0123] The experiments which relate to the identification and
characterization of a soluble gH-gL complex are now described.
[0124] The materials and methods used in these experiments are
presented below.
[0125] Cells and Viruses Mouse Ltk.sup.- cells (L cells) were
propagated in .alpha.-minimal essential medium (Gibco) supplemented
with 10% heat-inactivated fetal calf serum (FCS, obtained from
HyClone Laboratories), gentamicin, amphotericin B, vitamins, and
N-2-hydroxyethypiperazine-N'-2-ethanesulfonic acid (HEPES) buffer
solution. HSV-1 strain NS was propagated as described (Cines et
al., 1982, J. Clin. Invest. 69:123).
[0126] Antibodies Polyclonal antibodies and MAbs used in this study
were as follows: rabbit polyclonal anti-gH antibody preparations
R82 and R83 (Roberts et al., 1991, Virology 184:609); anti-gH MAbs
include 37S (Showalter et al., 1981, Infn. Immun. 34:684), 53S
(American Type Culture Collection), and LP11 (Buckmaster et al.,
1984, Virology 139:408); rabbit antiserum raised against the gL
UL1-2 peptide (anti-gL serum; Hutchinson et al., 1992, J. Virol.
66:2240).
[0127] Plasmids expressing gH Plasmid pCMV3gH-1 contains a 3.1 kb
HindIII fragment obtained from pSR95 (Roberts et al., 1991,
Virology 184:609), which fragment contains the entire HSV-1 strain
NS gH coding region ligated into the HindIII site in the polylinker
of pCMV3 (Andersson et al., 1989, J. Biol. Chem. 264:8222). Thus,
plasmid pCMV3 gH-1 encodes gH under the control of the human
cytomegalovirus immediate early promoter. A second plasmid,
pCMV3gH(792) encodes gH-1(792) which is a truncated form of the
wild type protein terminating at amino acid 792 and was constructed
by insertion of an SpeI linker containing termination codons within
the gH coding region as described by Roberts et al. (1991, Virology
184:609).
[0128] Plasmids expressing gL The UL 1 open reading frame which
encodes gL (McGeoch et al., 1988, J. Virol. 62:1486) was amplified
from viral DNA using the polymerase chain reaction (PCR). The
following synthetic oligonucleotide primers containing the
underlined XbaI restriction enzyme sites were designed to
facilitate cloning:
[0129] 5'-TGCTCTAGAGCGCTATGGGGATTTTGGGT-3' (upstream primer)
and
[0130] 5'-TGCTCTAGAGGTTTCCGTCGAGGCATCGT-3' (downstream primer)
[SEQ. ID. NOS; 1 and 2, respectively]. To prepare the template for
amplification, approximately 10.sup.5 HSV-1 virions were lysed by
heating to 95.degree. C. for 10 minutes. The lysate was added to a
100 .mu.l PCR reaction mixture containing 2.5 units of TaqI DNA
polymerase (Perkin Elmer, Cetus), 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl.sub.2, 0.001% gelatin (wt/vol), the four
deoxyribonucleotides each at a concentration of 200 .mu.M, and 1.0
.mu.M of each of the primers. The PCR mixture was subjected to 35
cycles of amplification (94.degree. C. for 1 minute, 55.degree. C.
for 1 minute, and 72.degree. C. for 1.5 minutes). The product, a
718 bp fragment was digested with XbaI in order to generate
cohesive termini and was then gel purified and ligated into the
XbaI sites of both pCMV3 (Andersson et al., 1989, J. Biol. Chem.
264:8222) and pMMTV (Friedman et al., 1989, Mol. Cell. Biol.
9:2303) generating the plasmids pCMV3gL-1 and pMMTVgL-1,
respectively. Transcription of the gL gene is thus under the
control of the CMV immediate early promoter in pCMV3gL-1 and under
the control of the inducible dexamethasone mouse mammary tumor
virus promoter in pMMTVgL-1.
[0131] Transfection of cells Transient transfections were performed
using calcium phosphate (Graham and van der Eb, 1973, Virology
52:456). In co-transfection assays, 4 .mu.g/well of plasmid was
used; for single plasmid transfections, 8 .mu.g/well was used. At
42 hours post-transfection, cell supernatants were collected and
assayed by immunoprecipitation and/or cells were harvested for
immunofluorescence studies.
[0132] Immunoprecipitation and gel electrophoresis of proteins At
18 hours post-transfection, L cells transfected with plasmid DNA as
described above were washed twice in Dulbecco's modified Eagle
medium lacking cysteine (DMEM/cys-; Gibco BRL). The cells were
incubated in DMEM/cys- supplemented with 200 .mu.Ci per well of
.sup.35S-cysteine and 10% FCS for 24 hours. Cell supernatants were
collected, centrifuged to remove any non-adherent cells and
concentrated 10-fold by centrifugation at 5520.times.g for 1 hour
in Centricon-10 concentrator tubes (Amicon, Inc.). Concentrated
supernatants were treated with 1 mM each of
N.alpha.-p-tosyl-L-lysine chloromethyl ketone (TLCK) and
N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and were stored
at -20.degree. C. For immunoprecipitation, supernatants were thawed
and mixed with a buffer containing 10 mM Tris-HCl, pH 8.0, 100 mM
NaCl, 1 mM EDTA. 0.5% Noniodet P-40 (wt/vol), and 0.25% gelatin
(wt/vol). Supernatants were incubated with 3 .mu.l of either R83 or
anti-gL serum and PANSORBIN Staphylococcus aureus cells
(CalBiochem). Following precipitation, immunoprecipitates were
washed three times in high salt buffer (10 mM phosphate buffer, pH
7.2, containing 0.65 M NaCl, 1 mM EDTA, 1% Triton X-100) and once
in low salt buffer (10 mM phosphate buffer, pH 7.2, containing 0.15
M NaCl, 1 mM EDTA, 1% Triton X-100). The immunoprecipitates were
then solubilized in dissolution buffer (100 Tris HCl, pH 6.8, 4%
SDS, 0.2% bromophenol blue, 20% glycerol and 10%
.beta.-mercaptoethanol), and the proteins were resolved by SDS-PAGE
under denaturing conditions as described (Cohen et al., 1986, J.
Virol. 60:157). Proteins in the gel were fixed in a mixture of
glacial acetic acid and methanol, the gel was impregnated with
Autofluor (National Diagnostics) and was exposed to X-ray film at
-70.degree. C.
[0133] Enzymatic treatment of cytoplasmic extracts and
immunoprecipitates Cytoplasmic extracts were prepared from infected
or transfected cells as described (Cohen et al., 1986, J. Virol.
60:157). Extracts were treated with 5 mU of
endo-.beta.-N-acetylglucosaminidase (endo H; Boehringer Mannheim
Biochemicals) or 250 mU of endoglycosidase F/N-glycosidase F (endo
F; Boehringer Mannheim Biochemicals) for 2 hours at 37.degree. C.
Immunoprecipitates were eluted from PANSORBIN cells by boiling in
buffer containing 0.1 M sodium phosphate (pH 7.5), 0.5%
.beta.-mercaptoethanol and 0.1% SDS, and were diluted 2-fold in 0.1
M sodium phosphate buffer containing 1% octyl glucoside, 150 .mu.M
phenanthroline and 10 mM EDTA. Samples were then treated for 2
hours at 37.degree. C. with 5 mU of endo H or 250 mU of endo F.
[0134] Western blot analysis Following enzymatic treatment of
cytoplasmic extracts, proteins were resolved by SDS-PAGE under
denaturing conditions and transferred to a nylon membrane which was
incubated in the presence of R82. Bound antibody was detected using
goat anti-rabbit horseradish peroxidase conjugate (Boehringer
Mannheim Biochemicals) and a chemiluminescent substrate solution
(New England Nuclear). The membrane was exposed at room temperature
to Kodak X ray film.
[0135] Immunofluorescence To examine expression of gH and gL on the
cell surface, cells transfected with plasmids expressing either
protein were suspended in phosphate-buffered saline (PBS)
containing 0.005 M EDTA, washed by centrifugation and resuspended
in PBS containing 1% bovine serum albumin (BSA). The cells were
incubated at 4.degree. C. for 30 minutes in the presence of gH MAb
alone, anti-gL-1 serum alone, or in the presence of both antibodies
(for double label immunofluorescence studies). The cells were
washed and further incubated in the presence of goat anti-mouse IgG
F(ab').sub.2 fluorescein-labeled conjugate (to detect gH) and/or
goat anti-rabbit IgG F(ab').sub.2 fluorescein-labeled or
rhodamine-labeled conjugate (to detect gL). Cells so stained were
visualized using a Leitz epifluorescence microscope. For
double-label fluorescence studies, the same microscopic fields were
viewed under fluorescein and rhodamine filters.
[0136] Additional immunofluorescence studies were performed on
acetone fixed cells. Transfected cells suspended in PBS containing
1% BSA were allowed to adhere to glass microscope slides and were
fixed in acetone prior to incubation in the presence of antibody
and conjugate as described above.
[0137] The results of these experiments are now described.
[0138] Requirement of gL for normal processing of gH in transfected
cells Normal processing of gH requires addition of N-linked
carbohydrates to the nascent molecule (Buckmaster et al., 1984,
Virology 139:408). However, in the absence of other HSV-1
glycoproteins, processing of gH is incomplete. In order to
establish a definitive role for gL in processing of gH, gH produced
in the presence or absence of gL was analyzed for sensitivity to
endo H or endo F. Treatment of incompletely processed glycoproteins
with either enzyme results in cleavage of the carbohydrate moiety
and a subsequent reduction in the molecular weight of the
glycoprotein compared with the completely processed glycoprotein.
Glycoproteins which contain complex, fully processed carbohydrate
moieties are resistant to cleavage by endo H, but remain sensitive
to endo F. Cytoplasmic extracts were prepared from cells which were
either infected with HSV-1 or were cotransfected with pCMV3gH-1 and
pCMV3gL-1, or simply transfected with pCMV3gH-1 alone, which
extracts were either subsequently untreated or were incubated in
the presence of endo H and endo F. It is evident from the data
presented in FIG. 1 that gH exhibits sensitivity to both endo H and
endo F when expressed in the absence of gL. In contrast, when gL is
present either during infection or during cotransfection, the
molecular weight of gH following endo H treatment is essentially
unchanged indicating that gL is required for complete processing of
gH in transfected cells.
[0139] Expression of gL is required for correct folding of gH To
determine whether gH is folded correctly in gL-negative cells,
reactivity of gH with the conformation-dependent gH-MAb, 53S, was
examined by immunofluorescence. Extracts of L cells transfected
with pCMV3gH-1 did not react with 53S (FIG. 2A), whereas extracts
of cells transfected with both pCMV3gH-1 and pCMV3gL-1 exhibited
strong reactivity (FIG. 2B). Similar results were observed using
LP1 1, a gH MAb which reacts with a distinct conformational
phenotype of gH (Gompels et al., 1991, J. Virol. 65:2393). These
results indicate that, in transfected cells, folding of gH is
normal in the presence of gL but is abnormal in the absence of
gL.
[0140] Expression of gL is required for intracellular transport and
cell surface expression of gH in transfected cells To examine the
effect of expression of gL on intracellular transport and cell
surface expression of gH, L cells were transfected with pCMV3gH-1
either alone or in combination with pCMV3gL-1. The intracellular
localization of gH in cells so transfected was assessed by
immunofluorescence using the MAb 37S, a MAb which binds to gH
irrespective of its structural conformation (Roberts et al., 1991,
Virology 184:609). In the absence of gL, the intracellular
distribution of gH was cytoplasmic and included some perinuclear
localization suggesting retention of this glycoprotein in the
endoplasmic reticulum (FIG. 3A). However, in the presence of gL, gH
was distributed throughout the cell in a uniform manner with some
localization at the cell perimeter suggesting cell surface
expression (FIG. 3B). To examine cells surface expression in more
detail, these studies were repeated using unfixed cells. In this
instance, gL was expressed from the dexamethasone-inducible plasmid
pMMTVgL-1. Thus, cells were cotransfected with pCMV3gH-1 and
pMMTVgL-1 and were subsequently incubated either in the presence or
absence of dexamethasone. Cells so transfected were treated with
the gH MAb 37S, and with anti-gL serum, and were then stained with
fluorescein-labeled conjugate (to detect gH) and rhodamine-labeled
conjugate (to detect gL). In cells incubated in the presence of
dexamethasone, both gH and gL co-localized to the cell surface
(FIG. 4A and FIG. 4B). This was also true when cells were
cotransfected with pCMV3gH-1 and pCMV3gL-1 (FIG. 4E and FIG. 4F).
However, in the absence of dexamethasone, neither glycoprotein was
found at the cell surface. Thus, gL is required for intracellular
processing of gH.
[0141] Membrane association of gL results from its association with
gH The predicted amino acid sequence of gL suggests that it is a
secreted rather than a membrane associated glycoprotein (McGeoch et
al., 1988, J. Gen. Virol. 69:1531). To investigate whether gL is
capable of independent association with the cell membrane (i.e., in
the absence of membrane association by gH), gL was co-expressed
with a mutant of gH, which mutant lacks the membrane spanning
domain of the glycoprotein (gH792). In cells transfected with a
plasmid encoding gL, or with a plasmid encoding gL and the
truncated form of gH, gL was not detected on the cell surface (FIG.
5B and FIG. 5D); however, in cells transfected with plasmids
encoding gL and full length wild type gH, gL was detected on the
cell surface (FIG. 5F). That gL was actually expressed in each of
these sets of transfected cells was confirmed by immunofluorescence
of permeabilized cells (FIG. 5A, FIG. 5C and FIG. 5E). These data
demonstrate that cell surface expression of gL is dependent upon
expression of wild type gH.
[0142] Secretion of gL To examine secretion of gL from transfected
cells, cells were transfected with the following combinations of
plasmids: pCMV3gL-1 alone; pCMV3gL-1 plus pCMV3gH(792); or,
pCMV3gL-1 plus pCMV3gH-1. In each instance, proteins synthesized by
these cells were labeled with .sup.35-cysteine, they were extracted
from the cell supernatants and were analyzed by immunoprecipitation
using anti-gL serum. A 30 kDa protein (gL) was identified in
supernatants from cells transfected with pCMV3gL-1 alone and in
cells transfected with pCMV3gL-1 plus pCMV3gH(792). This protein
was not identified in supernatants from cells cotransfected with
pCMV3gL-1 and pCMV3gH-1 (FIG. 6). A protein of 105 kDa in size was
also immunoprecipitated by anti-gL serum and by R83 (anti-gH MAb)
in cells cotransfected pCMV3gL-1 plus pCMV3gH(792) suggesting that
both gL and the truncated form of gH are secreted from these cells
as a complex (FIGS. 6 and 7). Neither the 30 nor the 105 kDa
proteins were evident in supernatants from cells transfected with
pCMV3gH(792) alone. Thus, these experiments demonstrate that gL and
gH are capable of forming a complex at the cell surface and that gL
does not independently associate with the cell membrane, rather,
its association with this membrane is dependent upon the presence
of the membrane anchor portion of gH. The finding that gL is
secreted from cells independent of gH was unexpected since previous
studies suggested that processing of gL required the presence of gH
(Hutchinson et al., 1992, J. Virol. 66:2240).
[0143] Confirmation that gH is not required for processing of the
carbohydrate moiety of gL To further investigate the role of gH in
processing of gL, cells were transfected with either pCMV3gL-1
alone or were cotransfected with pCMV3gL-1 and pCMV3gH(792).
Supernatants were collected from cells so transfected and any gL
present therein was examined for sensitivity to either endo H or
endo F. The same pattern of enzyme sensitivity was evident
irrespective of the presence or absence of gH (FIG. 8). Therefore,
gH is not required for either the addition or processing of
N-linked carbohydrates on gL expressed in transfected cells.
[0144] The region of gH required for complex formation with gL As
described above, coexpression of gL with the truncated form of gH
[gH(792)] results in a complex comprising the two proteins, which
complex is secreted from cells. To determine which domains of gH
are required for complex formation, additional mutants of gH,
expressing further truncated forms of this glycoprotein, were
tested in the transfection assay described above. The mutants
tested were as follows: gH(648), gH(475), and gH(102), each of
which expresses a protein of 648, 475 and 102 amino acids in
length. The plasmids encoding these mutated forms of gH are
designated pSR124 (648), pSR123 (475) and pSR125 (102). Each
plasmid encodes the truncated form of gH under the control of the
Rous sarcoma virus promoter (Roberts et al., 1991, Virology
184:609). Cells were cotransfected with pCMV3gL-1 and with one of
the mutant plasmids described above and were incubated in medium
containing .sup.35S-cysteine. At 18 hours post-transfection, cell
supernatants were harvested and the proteins contained therein were
immunoprecipitated with anti-gH serum, as described above.
Immunoprecipitates were resolved by SDS PAGE and the results are
shown in FIG. 9. Mutant forms of gH which terminate at amino acid
residues 792, 648 and 475 were secreted from cotransfected cells in
a complex with gL. However, when a mutant encoding only 102 amino
acid residues of gH was used, a gH-gL complex was not detected in
cell supernatants. Additional experiments have been conducted in a
similar manner to that described above and it is now believed that
a region of gH comprising amino acids 1-324 and a region comprising
amino acids 1-275 are capable of forming a complex with gL. Thus,
the region of gH required for interaction with gL resides in the
amino-terminal portion of the molecule between residues 1-275. As
noted above, the membrane anchor region of gH resides in the
carboxyl terminus of the molecule between amino acid residue 792
and the last amino acid residue at the carboxyl terminus.
[0145] Construction of a stable RH-gL expressing cell line For the
purposes of vaccine production, generation of a gH-gL complex in
the cell lines described below has significant advantages over
other methods of production of this complex which methods may
involve for example, extraction of a gH-gL complex from infected
cells. In the latter case, since wild type gH comprises a
hydrophobic membrane anchor region, it is necessary to use
detergents during extraction to remove the membrane portion. Such
treatment may in fact alter the conformation of the complex and
thereby alter its immunogenic properties. By using the cell lines
described below, a secreted form of gH is produced as a complex
with gL and thus, further extraction and purification prior to use
as a vaccine is minimized. In addition, the use of mammalian cells
is advantageous in that both insect and lower eukaryotic cells each
process carbohydrates somewhat differently than do mammalian cells.
Thus, the use of mammalian cells ensures correct processing of the
components of the complex and thereby ensuring preservation of the
immunologically protective epitopes within the complex.
[0146] A diagram of the plasmids used to generate a gHt-gL
expressing cell line, and a map of gH and gL is provided in FIG.
10.
[0147] Cell lines which constitutively express and secrete gH-gL as
a complex were constructed as follows. L cells were cotransfected
with pCMVgH(792) and pCMVgL-1 and with the plasmid pX343 which
encodes a gene conferring resistance of cells to hygromycin B
(Blochlinger et al., 1984, Mol. Cell. Biol. 4:2929). Cells so
transfected were incubated in the presence of hygromycin B (200
.mu.g/ml). Twenty four clones of hygromycin B resistant cells were
selected and supernatants therefrom were first screened for
production of gH by Western blot analysis. Of these, two clones
were further tested for production of both gH and gL by
immunoprecipitation. Both of these clones expressed and secreted
the gH-gL complex one of these clones designated HL-7, produced
large amounts of the complex and was therefore selected for
additional studies.
[0148] The gH-gL complex secreted by HL-7 cells was found to be
immunoprecipitable by gH MAbs 52S, 53 S, and LP 11 (FIG. 11). Since
these MAbs are dependent upon correct folding of gH in that they
react with distinct structural epitopes on the molecule, the gH-gL
complex secreted by HL-7 cells appears to have a structure similar
to that of the wild type complex.
[0149] Identification of a putative membrane fusion region of gH A
computer-based analysis of the amino acid sequence of gH was
performed and revealed a region of the protein predicted to form an
amphophilic .alpha.-helix extending from approximately amino acid
residues 280-310, which helix is indicative of a membrane fusion
region in influenza virus (White, 1992, Science 258:917). A gH
expression plasmid (derived from pCMV3gH-1) was constructed wherein
amino acid residues 275-324 were deleted (i.e., the deleted amino
acid residues encompass the putative membrane fusion region) and a
linker encoding 5 amino acids was inserted at the site of the
deletion to re-establish the correct reading frame. The resulting
gH mutant, pCMVgH.DELTA.(275-324), encodes a protein which folds
correctly and is transported to the cell surface when expressed in
cells also expressing wild type gL. Furthermore, this deleted gH
retains the ability to form a complex with gL as assessed in the
co-immunoprecipitation assay described above. The ability of the
deleted gH to rescue a gH negative virus was determined in a
complementation assay as follows. L cells were transiently
transfected with pCMVgH-1 or pCMVgH.DELTA.(275-324), or were mock
transfected. At 18 hours post-transfection, cells were infected
with a gH negative mutant virus and incubation was continued for an
additional 24 hours. The amount of virus produced was then assessed
by plaque assay. Cells transfected with pCMVgH-1 produced
2.5.times.10.sup.5 infectious virus per ml while mock transfected
cells produced less than 1.5.times.10.sup.2 virus per ml. Cells
which were transfected with pCMVgH.DELTA.(275-324) also produced
negligible amounts of infectious virus i.e., less than
1.5.times.10.sup.2 virus per ml, indicating that this plasmid was
incapable of rescuing the gH negative phenotype exhibited by the gH
negative virus. Thus, deletion of gH in the region of amino acid
residues 275-324 renders the protein non-functional.
EXAMPLE 2
[0150] The experiments which establish that gH-gL complex functions
to neutralize virus infectivity and protect animals against
challenge by wild type infections virus are now described. The
Materials and Methods used in these experiments are presented
below.
[0151] Cells and virus African green monkey kidney (Vero) and mouse
L cells were grown in Dulbecco's Modified Eagle Medium (DMEM)
supplemented with 5% fetal bovine serum (FBS) at 37.degree. C. D14
cells (Vero derived) which express HSV-1 ICP6 (Goldstein et al.,
1988, J. Virol. 62:2970-2977) were grown in DMEM with 5% FBS and
G418 (25 .mu.g/ml) at 37.degree. C. HL-7 cells which express gHt-gL
were grown in DMEM supplemented with 10% FBS and hygromycin B (50
.mu.g/ml). For protein production, hygromycin B was eliminated from
the medium. HSV-1(hrR3) (Goldstein, supra) was propagated on D14
cells and titered on Vero cells. The propagation of HSV-1 (NS) and
HSV-2 (333) virus stocks have been described (Eisenberg et al,
1987, Microb. Pathog. 3:423-435).
[0152] Antibodies The monoclonal antibody (MAb) secreting cell
lines, 52S and 53S (recognizing gH-1) (Showalter et al., 1981,
Infect. Immun. 34:684-692) were obtained from the American Type
Culture Collection. Anti-gH MAb 37S is described in Showalter et
al., supra). Anti-gH MAb LP11 is described in Buckmaster et al.
(1984, Virology 139:408-413). MAb 8H4, which recognizes a linear
epitope on gL is described herein and in Dubin et al. (1995, J.
Virol. 69:4564-4568); Rabbit antibody .alpha.UL1-2, which was
prepared against a peptide sequence of gL is described in
Hutchinson et al. (1992, J. Virol. 66:2240-2250). Rabbit antibody
R83 (against gH) is described herein and in Roberts et al. (1991,
Virology 184:609-624). Polyclonal antibodies R137, R138, R139 and
R140 were prepared against purified gHt-gL as described herein.
[0153] Purification of gHt-gL complex from the supernatant of HL-7
cells The construction of the HL-7 cell line has been described
herein. To obtain gHt-gL complex from HL-7 cells, the cells were
grown in roller bottles. The supernatant was obtained after 3 days
and replaced with fresh medium. Two "harvests" of supernatant were
obtained from each roller bottle.
[0154] The secreted gHt-gL complex was purified by chromatography
on an immunoaffinity column of 53S, a gH-1 specific MAb, by a
modification of a previously method used to purify gH from extracts
of HSV-1 infected cells (Roberts et al, 1991, Virology
184:609-624). In the present experiment, the clarified medium was
passed over the column, and the bound protein was eluted with a low
pH buffer consisting of 50 mM glycine, 0.5 M NaCl, pH 2.5. The
eluate was neutralized with 1 M Tris-base (pH 9.0) and was
concentrated. Protein was quantitated by using the BCA kit
(Pierce). Approximately 400 .mu.g of gHt-gL complex was obtained
per liter of HL-7 cell supernatant (approximately 10.sup.-4
ng/cell).
[0155] To quantitate the amount of complex obtained in the
concentrated samples, the intensity of staining of the gH and gL
bands on SDS-PAGE was compared with known quantities of protein
standard (bovine serum albumin). These data are presented in FIG.
12. It is estimated that each sample contained approximately 0.5
.mu.g/.mu.l of gH-gL complex. It is possible to purify 0.5 mg of
gH-gL complex from approximately 1 liter of HL-7 supernatant using
the procedures described herein. Further, densitometric analysis of
the silver stained gel indicates that gH and gL are present in the
complex at a purity of approximately 90%. In addition,
approximately 50% of the gH-gL complex secreted from HL-7 cells
possess a conformation which is indistinguishable from that of the
native molecule as assessed by binding to the 53S MAb.
[0156] Purification of HSV-1 Virus was purified as described in
Handler et al. (1996, J. Virol. 70:6067-6075). Briefly, roller
bottles (850 cm.sup.2 ) of D14 cells were infected with hrR3 at an
MOI of 0.1. The growth medium was collected at 24 h post infection
and extracellular virus was pelleted by centrifugation at
100,000.times.g through a 5% sucrose-PBS cushion. Virus was further
purified by first resuspending the pellet in PBS, followed by
centrifugation at 30,000.times.g for 5 hours through a 10-30-60%
sucrose-PBS step gradient. The virus band located at the 30-60%
sucrose interface was collected, titered and stored at -80.degree.
C.
[0157] Soluble HSV glycoproteins and infected cell extracts Soluble
gD1(306t) was produced in baculovirus infected Sf9 cells and was
purified as described (Sisk et al., 1994, J. Virol. 68:766-775).
Cytoplasmic extracts of HSV-1 (NS) (Friedman et al., 1984, Nature
(London) 309:633-635) or HSV-2 (333) infected cells were prepared
as described (Eisenberg et al., 1987, Microb. Pathog. 3:423-435;
Eisenberg et al., 1982, J. Virol. 41:1099-1104). Full length gD-1
was purified from cytoplasmic extracts of HSV-1 infected cells
(Eisenberg et al., 1987, Microb. Pathog. 3:423-435). Soluble
gC-1(457t) was produced from baculovirus infected insect cells and
was purified as described (Tal-Singer et al., 1995, J. Virol.
69:4471-4483).
[0158] SDS-PAGE and Western blot analysis SDS-PAGE under denaturing
or "native" conditions was performed as described in Cohen et al.
(1986, J. Virol. 60:157-166), using Tris-Glycine 10% or 4-12%
gradient precast gels (Novex Experimental Technology). Silver
staining was performed-using a silver staining kit (Pharmacia
Biotech). For Western-blot analysis, proteins were transferred to
nitrocellulose, probed with anti-serum R83 for gH or MAb 8H4 for
gL. Goat anti-rabbit (in the case of R83) or anti-mouse (in the
case of 8H4) IgG-Peroxidase (Boehringer) was then added as
secondary antibody and bands were visualized on X-ray film after
the addition of ECL chemiluminescent substrate (Amersham). To strip
the blot, 50 mM glycine, 0.5 M NaCl, pH 2.5 was added and the blot
was incubated at room temperature for 15 minute. The blot was then
washed with PBS-0.2% Tween and was reprobed.
[0159] Antigenic analysis of gHt-gL by ELISA Various concentrations
of gHt-gL were coated onto ELISA plates and incubated overnight at
4.degree. C. The plates were blocked with PBS containing 1% bovine
serum albumin (B SA) and 1% ovalbumin (OVA). MAbs LP1 1, 53S and
37S were each diluted in PBS containing 0.05% BSA and 0.05% OVA and
were then added to the ELISA plate to detect the presence of gH.
After 1 hour at room temperature, the plate was washed three times
with PBS-0.5% Tween 20. Goat-anti-mouse-IgG-horseradish peroxidase
conjugate (Boehringer) was added and the plate was incubated at
room temperature for 30 minutes. After a rinse with citrate buffer
(20 mM citrate acid, pH 4.5), ABTS substrate
(2,2'-azino-di-3-ethylbenzthiozoline-6-sulfonic acid, Moss, Inc.)
was added and absorbance was read at 405 nm using a microtiter
plate reader (Biotek).
[0160] HSV-1 entry assay Vero cells were seeded onto a 96 well
plate and grown to confluence. The plate was cooled at 4.degree. C.
for 10 minutes, and viral glycoproteins which had been serially
diluted in 5% FBS DMEM (with 0.03 M HEPES) were added. The medium
was removed and replaced with 50 .mu.l of a single purified virion
glycoprotein and incubated at 4.degree. C. for 90 minutes. Purified
HSV-1(hrR3) in 5% FBS DMEM (2.times.10.sup.4 PFU/ml) was added to
each well (the MOI was 0.5 PFU/cell) and the wells were incubated
at 4.degree. C. for 90 minutes to allow virus to attach to the
cells. The cells were then incubated for 5 hours at 37.degree. C.
and were lysed with 1% NP-40 in DMEM. 50 .mu.l of lysate obtained
from each well was transferred to an ELISA plate, mixed with 50
.mu.l CPRG (4.8 mg/ml) (Chlorophenolred-.beta.-D-galactopyranosid-
e, Boehringer) and .beta.-galactosidase activity was measured by
measuring the absorbance at 570 nm every 2 minutes for a total of
25 measurements using an ELISA plate reader (Bio-Tek). The slope of
the line was used to calculate the amount of .beta.-galactosidase
activity as mOD/min.
[0161] Immunization of Rabbits with gHt-gL New Zealand rabbits were
immunized with gHt-gL mixed with one of two adjuvants (Set I and
Set II) as follows. Rabbits in Set I were immunized with gHt-gL
(150 .mu.g total) mixed with Freund's adjuvant. The first dose was
in Freund's Complete adjuvant (Sigma) and subsequent injections
were given in Freund's incomplete adjuvant. Rabbits in Set II were
immunized with gHt-gL mixed with an equal volume of Alum adjuvant
(Pierce Chemical Co.).
[0162] Virus neutralization assay Rabbit or mouse sera were treated
at 56.degree. C. for 30 minutes to inactivate complement. Serial
two fold dilutions of serum were prepared in DMEM containing 5%
FBS. The serum was then mixed with an equal volume of HSV-1 or
HSV-2 adjusted to yield 100 plaques per well in the absence of
neutralizing antibody. The virus cell mixture was incubated for 1
hour at 37.degree. C., and was then overlaid with medium and was
incubated at 37.degree. C. for 24 hours. The medium was removed,
the cells were fixed in a 2:1 mixture of methanol and acetone and
were dried. Plaques were visualized using a cocktail of polyclonal
antibodies to gD, gB and gC, by "black plaque assay" (Highlander et
al., 1987, J. Virol. 61:3356-3364; Tal-Singer et al., 1995, J.
Virol. 69:4471-4483) using horseradish peroxidase conjugated
protein A, followed by addition of the substrate
4-chloro-1-naphthol. The neutralization titer was expressed as the
dilution of serum that reduced the number of plaques by 50%.
[0163] Two assays were used to measure serum blocking
(neutralization) of virus entry. In the first (antibody+virus
method), each antiserum was mixed with 4.times.10.sup.5 PFU/ml hrR3
in DMEM containing 5% FBS, and 0.03 M HEPES, and the serum-virus
mixture was incubated at 37.degree. C. for 90 minutes, cooled to
4.degree. C. and added to Vero cells in 96 well plates in a volume
of 100 .mu.l/well. Plates were rocked at 4.degree. C. for 90
minutes, then shifted to 37.degree. C. for 5 hours. Cells were
lysed and .beta.-galactosidase activity was measured in the
cytoplasmic extract. In the second assay (antibody after virus
method), 4.times.10.sup.5 PFU/ml of hrR3 in DMEM containing 5% FBS
and 0.03 M HEPES was added to Vero cells at 4.degree. C. for 90
minutes. The virus was removed and replaced by antiserum diluted in
DMEM containing 5% FBS. Plates were rocked at 4 .degree. C. for 90
minutes, then shifted to 37.degree. C. for 5 hours. Cells were
lysed and .beta.-galactosidase activity was measured.
[0164] Murine flank(zosteriform) model of HSV Challenge A
zosteriform model of HSV-1 infection (Simmons et al., 1985, J.
Virol. 53:944-948; Simmons et al., 1984, J. Virol. 52:816-821) was
used to test the efficacy of gHt-gL as a vaccine. Nine to ten week
old Balb/c (Charles River) mice were immunized intraperitoneally
with 10 .mu.g antigen in complete Freund's adjuvant, followed by
three additional 10 .mu.g doses of antigen given in incomplete
Freund's adjuvant at two week intervals. The antigens used were
purified gHt-gL produced by HL-7 cells, or purified full length
gD-1 purified from HSV-1 infected cells (Eisenberg et al., 1987,
Microb. Pathog. 3:423-435). Sham-immunized control animals received
PBS emulsified with adjuvant at the same intervals. Mice were bled
and sera were obtained therefrom between the third and fourth
immunizations to test for virus neutralization. Two weeks after the
last immunization, the right flank of each immunized or control
animal was shaved and denuded using a dipilatory cream. Twenty four
hours later, 5.times.10.sup.5 PFU of HSV-1 was applied to the
depilated flank approximately 3 mm ventral to the spinal column and
the skin was scratched with a 27 gauge needle using 20 horizontal
strokes and 20 vertical strokes over an approximate area of
3.times.3 mm. The flank was observed daily for at least 10 days and
cumulative scores for primary and secondary areas were recorded for
days 3 through 8. The period of recording lesions was limited to
this period due to the deaths of unprotected animals beginning at
day 8. Disease at the inoculation site was scored as follows: 0
points for no disease; 0.5 for swelling without vesicles; and 1
point each for each vesicle or scab to a maximum score of 5.
Swelling and lesions in locations separate from the inoculation
site were considered to be secondary or zosteriform disease.
Scoring of these lesions was the same as for the inoculation site
except that a daily maximal score of 10 was used.
[0165] The results of these experiments are now described.
[0166] HL-7 cells secreted significant amounts of gHt-gL which was
detected on western blots at the predicted sizes for gHt and gL as
described herein.
[0167] Purification and analysis of gHt-gL gHt-gL was purified from
the growth medium of HL-7 cells by immunoaffinity chromatography on
an anti-gH(53S) MAb column as described herein. Purification was
monitored by SDS-PAGE followed by silver staining (FIG. 13A) as
well as by western blot analysis (FIG. 13B, FIG. 13C). The purified
complex contained two silver stained bands of 110 kDa and 35 kDa in
size (FIG. 13A, lane 3), although neither of these glycoproteins
was prominent in the culture supernatant or column flow through
(FIG. 13A, lanes 1 and 2). Both (FIG. 13B, FIG. 13C, lanes 1). The
absence of both gH and gL from the column flow through fraction
(FIG. 13B, FIG. 13C, lanes 2) established that all of the secreted
gL was associated with gH and both proteins bound to MAb 53S as a
stable complex. Both proteins were eluted by low pH (FIG. 13B, FIG.
13C, lanes 3). It was estimated that the eluted complex was greater
than 95% pure by silver stain (FIG. 13A, lane 3).
[0168] LP11 reactivity is considered to be a critical test of gH-gL
conformation, since this MAb only reacts with gH when it is part of
the native complex (Hutchinson et al., 1992, J. Virol.
66:2240-2250). Secondly, LP1 1 neutralizes virus infectivity at
high titers and therefore recognizes an immunologically important
epitope (Buckmaster et al., 1984, Virology. 139:408-413). Purified
gHt-gL was immunoprecipitated using LP 11, separated by SDS-PAGE,
and analyzed by Western blotting, probing for gH (FIG. 14A, lane 1)
and gL (FIG. 14A, lane 2) on individual nitrocellulose strips. Both
proteins were detected establishing that the complex was reactive
with LP 11. Similar results were obtained using MAb 52S (Showalter
et al., supra) in the initial immunoprecipitation. In a second
method, ELISA was used to establish that the purified complex
reacts with MAbs LP1 1, 53S and 37S (Showalter et al., supra).
Previous studies established that MAbs 52S, 53S and LP1 1 recognize
different conformation dependent epitopes (Forrester et al., 1991,
J. Gen. Virol. 72:369-75; Fuller et al., 1989, J. Virol.
63:3435-3443; Fuller et al., 1989, J. Virol. 63:3435-3443; Roberts
et al., 1991, Virology. 184:609-624) and 37S recognizes a linear
epitope (Roberts et al., 1991, supra). Thus, these two experiments
indicate that gHt in the complex is antigenically correct. Similar
studies were not done on gL, as no conformation dependent MAbs are
available. However, the complex does react by ELISA with gL MAbs
which recognize linear epitopes.
[0169] gHt-gL does not inhibit virus entry Both gH-1 and gL-1 are
essential for HSV-1 penetration and cell-to-cell spread and are
most likely involved in a cell fusion event (Davis-Poynter et al,
1994, J. Virol. 68:7586-7590; Desai et al., 1988, J. Gen. Virol.
69:1147-56; Forrester et al., 1992, J. Virol. 66:341-348; Novotny
et al., 1996, Virology 221:1-13; Roop et al, 1993, J. Virol.
67:2285-2297). However, little is known about gH-gL function, or
whether the proteins work individually or together with other
glycoproteins to effect virus entry. Soluble forms of gD (gDt) are
able to block HSV infection (Johnson et al., 1990, J. Virol.
64:2569-2576; Nicola et al., 1997, J. Virol. 71:2940-2946; Nicola
et al., 1996, J. Virol. 70:3815-3822). This is due to the
interaction of gDt with cellular receptors such as HVEM (Montgomery
et al., 1996, Cell. 87:427-436; Whitbeck et al., 1997, J. Virol.
71:6083-6093), rendering these receptors unavailable for binding to
gD in the virion. In contrast, soluble forms of gC-1 (gC-1t) do not
block plaque formation by HSV (Tal-Singer et al., 1995, J. Virol.
69:4471-4483). It has recently been reported that gC-1t, gB-1t and
gHt-gL do not bind directly to HVEM (Whitbeck et al., 1997,
supra).
[0170] The question in the present study is whether soluble gHt-gL
is able to block HSV-1 entry into cells, perhaps by binding to a
different receptor than HVEM. To answer this, an entry assay
employing HSV-1(hrR3) which contains the lacZ gene under the
control of the ICP6 promoter was used (Goldstein et al., 1988, J.
Virol. 62:2970-2977). Virus entry was measured as an increase in
.beta.-galactosidase activity at 5 hours post-infection (FIG. 1
5A). As expected from previous studies (Tal-Singer et al., 1995,
supra), gC-1t did not block virus entry and served as a negative
control for the assay. Fifty percent inhibition of virus entry was
observed at 50 nM gD-1(306t), a result similar to that obtained
using a 50% inhibition of plaque formation assay (Tal-Singer et
al., 1995, supra). In contrast, gHt-gL did not inhibit virus entry
even at protein concentrations as high as 350 nM (50 ng/.mu.l).
[0171] To determine whether gHt-gL enhanced the ability of soluble
gD-1(306t) to block infection by enhancing its binding to HVEM or
other gD receptors, the following experiment was carried out.
Increasing amounts of gHt-gL were added to cells together with 40
nM gD (FIG. 15B). At this concentration, gD inhibited virus entry
by 40-50%. gHt-gL did not enhance the inhibition achieved with gDt
alone.
[0172] Antibodies to gHt-gL block virus entry and neutralize virus
infectivity entry. It was previously shown that anti-gH
neutralizing MAbs such as LP1 1 are able to block HSV infection
even when added after virus attachment (Fuller et al., 1989, J.
Virol. 63:3435-3443). It was therefore hypothesized that if the
conformation of gHt-gL is close to that of the functional form in
the virus, then antibodies to the complex should be able to
neutralize infection and block virus entry whether added before or
after virus attachment.
[0173] To test this hypothesis, rabbits were immunized with gHt-gL
using either Freund's or alum adjuvant. All four animals produced
antibodies which recognized gHt and gL on western blot of a
denaturing gels (FIG. 16A). On a western blot of a non-denaturing
(native) gel (Cohen et al., 1986, J. Virol. 60:157-166), these
antibodies also recognized higher molecular weight forms on (FIG.
16B), corresponding in size to oligomers of gHt-gL.
[0174] All four sera tested exhibited significant titers of
complement independent HSV-1 neutralizing activity (Table 1). In
addition these sera also neutralized HSV-2, albeit at a much
reduced potency. These results indicated that the immunizing
protein had biologic activity. In addition, each of the sera, when
premixed with hrR3 virus, was able to block virus entry (FIG.
17A).
1TABLE 1 HSV neutralizing activity of sera for rabbits immunized
with gHt-gL Virus Neutralization Titer (50%) Plaque Plaque Entry
assay.sup.a assay.sup.b assay.sup.b Adjuvant Rabbit HSV-1 HSV-1
HSV-2(333) Freund's R136 (prebleed) <1:20 <1:20 <1:20 R137
(prebleed) <1:20 <1:20 <1:20 R136 (3rd bleed) 1:640 1:640
<1:20 R137 (3rd bleed) 1:4500 1:2000 1.60 Alum R138 (prebleed)
<1:20 <1:20 <1:20 R139 (prebleed) <1:20 <1:20
<1:20 R138 (3rd bleed) 1:2560 1:1800 1:100 R139 (3rd bleed)
1:1280 1:640 1:100 .sup.aVirus entry was measured by infecting
cells with HSV-1 strain hrR3 (Goldstein et al., 1998, supra) and
measuring-galactosidase activity at 5 hours post infection. The
neutralization titer represents the dilution of antiserum needed to
reduce .beta.-balactosidase activity to 50% of the maxium seen with
no serum added. .sup.bVirus infection was measured by plaque
formation by HSV-1 strain KOS on Vero cells using the black plaque
assay. The neutralization titer represents the dilution of
antiserum needed to reduce the number of plaques to 50% of the
number found on control plates with no anterium added.
[0175] As a second approach, the virus was first adsorbed to cells
at 4.degree. C. and either R83 (anti-gH), R137, or MAb LP11
antibody was subsequently added to the virus-cell mixture. As
expected, LP11 blocked virus entry when added after virus
adsorption (FIG. 17B). R137 exhibited similar blocking activity to
that observed for LP11, indicating that both antibodies recognized
a site on gH-2L which was critical for post-binding steps in virus
entry. This experiment suggests that the gHt-gL complex used to
prepare R137 contains a functionally active conformation. In
contrast, R83 antibody was unable to block virus entry. This was an
important control for the present study because R83 had been
prepared against gH purified from infected cells in such a way that
it lacked gL and therefore lacked the proper biologically active
conformation. (Roberts et al., 1991, Virology. 184:609-624). Thus,
although it was not possible to directly demonstrate blocking
activity by gHt-gL, the data provide indirect evidence that the
complex contains the conformation necessary for function in virus
infection.
[0176] Immunization of mice with gHt-gL To further assess the
ability of gHt-gL to elicit a humoral immune response, Balb/c mice
were immunized with gHt-gL in two separate experiments (Table 2).
As positive controls, other mice were immunized with gD purified
from HSV infected cells, and as negative controls, animals were
sham-immunized with PBS. Prior to challenge with virus, serum
samples were obtained from each of the immunized animals. The
reactivity of a pool of mouse anti-gHt-gL serum (from Experiment I)
was compared to that of R137 by immunoblotting. Both R137 and the
mouse anti-gHt-gL reacted with gHt and gL on western blots (FIG.
17, lanes 1 and 5). R137 reacted with two bands of 66 kDa and 45
kDa in extracts from both infected and uninfected cells (FIG. 18,
lanes 2, 3 and 4). Therefore, these bands are considered to be
reacting non-specifically with the antibody. The reactivity of
rabbit and mouse sera against cytoplasmic extracts of HSV-1 and
HSV-2 infected cells was also compared. Both R137 and the pooled
mouse serum reacted against bands migrating at the expected
positions of gH-1 and gL-1 (FIG. 18, lanes 2 and 6). These sera
also recognized the precursor forms of gH and gL. Both sera
cross-reacted against bands presumed to be pgH-2 and gH-2 (FIG. 18,
lanes 3 and 7). The mouse serum also reacted with a band at the
presumed position of gL-2 (FIG. 18, lane 8). It should be noted
that gL-2 is expected to be 500 Da larger than gL-1 based on
predicted amino acid sequence and gH-2 is predicted to be 700 Da
smaller than gH-1.
2TABLE 2 Protection of mice from intradermal HSV-1 challenge
following immunization with gD or gHt-gL. Average 1.sup.0 Average
2.sup.0 Immunizing Score.sup.b Score.sup.c Experment.sup.a Antigen
(Sum of d3-d8) (Sum of d3-d8) I Mock (PBS) 10.5 16.5 gD 5.5 0
gHt-gL 2.8 0 II Mock (PBS) 22.3 19.6 gD 5.8 0 gHt-gL 4.2 0 .sup.aIn
experiment I, there were ten mice in each group. One mouse in the
gH-gL immunized group of experiment I died prior to challenge. Of
the ten PBS infected animals, five died after virus challenge. Of
the mice immunized with either gD or gH-gL, no mice died after
virus challenge. In experiment II, there were five mice in each
group. Of the five PBS infected animals, all died as a result of
virus challenge. Of the five mice immunized with either gD or
gH-gL, no mice died after virus challenge. .sup.bPrimary lesions
developed immediately around the infection site. The primary scores
were cumulative from day 3 to day 8. Score range was 0-5.
.sup.cScored by the zosteriform lesions on the flank ssurrounding
the primary infection site. The secondary scores with cumulative
from days 3 to 8. Score range was 0-10.
[0177] Sera obtained from each mouse immunized with either gD or
gHt-gL exhibited high titers of virus neutralizing activity as
measured by inhibition of virus entry (data for representative mice
are shown in FIG. 19). It was observed that the titers were
approximately tenfold higher when animals were immunized with gD as
opposed to gHt-gL.
[0178] gHt-gL protects mice from HSV-1 challenge A zosteriform
model of HSV-1 infection was used to examine the ability of gHt-gL
to act as a vaccine (Simmons et al., 1985, J. Virol. 53:944-948;
Simmons et al., 1984, J. Virol. 52:816-821). Following
intraperitoneal immunization with either gD or gHt-gL, mice were
challenged with HSV-1 by intradermal inoculation on the right flank
(Table 2). In two separate experiments, some of the animals in each
group exhibited some evidence of infection at the site of virus
challenge (primary lesions). However, the primary lesion scores for
mice immunized with either gD or gHt-gL were lower than those of
sham-immunized mice. Of most significance was the finding that all
of the sham-immunized mice that developed primary lesions went on
to develop severe secondary zosteriform lesions. In contrast, mice
which were immunized with either gD or with gHt-gL exhibited no
secondary lesions, regardless of whether they developed any
evidence of primary lesions. Furthermore, all of the immunized mice
survived virus challenge, while many of the control animals died
(5/10 in experiment I and 5/5 in experiment II). These results
suggest that gHt-gL purified from HL-7 cells is biologically active
and is a likely candidate for use as a subunit vaccine against
HSV-1 infection.
[0179] Although neither gD nor gHt-gL were able to completely
protect mice from developing lesions at the site of primary
inoculation, both protein preparations ameliorated the severity of
the primary disease. Significantly, prior immunization with either
gD or gHt-gL gave excellent protection against development of
zosteriform lesions. These data, together with the neutralization
titers of anti-gHt-gL sera, are the most encouraging results seen
to date regarding the potential efficacy of gHt-gL as a
vaccine.
[0180] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit of the invention.
The appended claims are intended to be construed to include all
such embodiments and equivalent variations.
Sequence CWU 1
1
4 1 6 PRT artificial sequence exemplary sequence 1 Cys Thr Ala Gly
Tyr Arg 1 5 2 6 PRT Artificial sequence Exemplary sequence 2 Cys
Thr Ala Cys Arg Tyr 1 5 3 29 DNA Artificial sequence PCR primer 3
tgctctagag cgctatgggg attttgggt 29 4 29 DNA Artificial sequence PCR
primer 4 tgctctagag gtttccgtcg aggcatcgt 29
* * * * *