Nov 12, 1992 - JAMES TARTAGLIA,' OSWALD JARRE1T,2 JAMES C. NEIL,2 PHILIPPE ..... Earl, P. L., B. Moss, R. P. Morrison, K. Wehrly, J. Nishio, and.
JOURNAL OF VIROLOGY, Apr. 1993, p. 2370-2375
Vol. 67, No. 4
0022-538X/93/042370-06$02.00/0 Copyright © 1993, American Society for Microbiology
Protection of Cats against Feline Leukemia Virus by Vaccination with a Canarypox Virus Recombinant, ALVAC-FL JAMES TARTAGLIA,' OSWALD
JARRE1T,2 JAMES C. NEIL,2 PHILIPPE DESMETTRE,3 AND ENZO PAOLETTIf*
Virogenetics Corporation, 465 Jordan Road, Rensselaer Technology Park, Troy, New York 121801; Department of Veterinary Pathology, University of Glasgow, Bearsden, Glasgow G61 IQH, United Kingdom2; and Rhone Merieux, 69342 Lyon Cedex 07, France3 Received 12 November 1992/Accepted 7 January 1993
Two ALVAC (canarypox virus)-based recombinant viruses expressing the feline leukemia virus (FeLV) subgroup A env and gag genes were assessed for their protective efficacy in cats. Both recombinant viruses contained the entire gag gene. ALVAC-FL also expressed the entire envelope glycoprotein, while ALVACFL(dl IS) expressed an env-specific gene product deleted of the putative immunosuppressive region. Although only 50% of the cats vaccinated with ALVAC-FL(dl IS) were protected against persistent viremia after oronasal exposure to a homologous FeLV isolate, all cats administered ALVAC-FL resisted the challenge exposure. Significantly, protection was afforded in the absence of detectable FeLV-neutralizing antibodies. These results represent the first effective vaccination of cats against FeLV with a poxvirus-based recombinant vector and have implications that are relevant not only to FeLV vaccine development but also to developing vaccines against other retroviruses, including human immunodeficiency virus.
istics to the vector in nonavian species (1, 24). Since the vector does not replicate, disseminated infection within the vaccinee or spread to contacts or the general environment should be eliminated. The highly attenuated character of ALVAC has been demonstrated in both immunocompetent and immunocompromised animal models (24). Recent phase I clinical trials of an ALVAC-rabies virus glycoprotein G
The canarypox virus-based vector ALVAC is restricted for productive replication to avian species (7). However, ALVAC recombinant viruses express extrinsic immunogens when inoculated into nonavian species without apparent replication of the vector virus itself and have been shown to elicit protective immune responses (26, 28). ALVAC's hostrestricted phenotype imparts naturally attenuated character-
ATG
1imiI g 1gIPRI
RO/ RT
env I
IN
I Ir
LTR I
ALVAC-FL
EI
ATG
gg9
TAA
IPR I
ATG
TAA
ATG
TAA
FH6r nT I
ALVAC-FL(d/ IS) ATG
iz ga99IgPR I
TAA
H-6f
OAis
I
aa315 I s aa341 FIG. 1. Schematic representation of the FeLV provirus and regions incorporated into ALVAC recombinant viruses. Architectural features (16) are designated by LTR (long terminal repeat), gag (group-specific antigens, including the major core protein, p27, and the nucleoprotein), pol (gene encoding protease [PR], reverse transcriptase [RT], and integrase [IN]), and env (gene encoding the precursor envelope glycoprotein p85, which is proteolytically cleaved to the p7O and plSE mature forms). Below the provirus structure are depicted thegag and protease (PR) regions and the env sequences used to generate poxvirus expression cassettes for insertion into the ALVAC vector. The FeLV-A env and gag genes were derived from PstI subclones of pFGA-2, described elsewhere (23). These genes were fused with the vaccinia virus H6 early, late promoter element (19). ALVAC-FL (vCP97) and ALVAC-FL(dl IS) (vCP93) contain the same gag expression cassette but differ in the env expression cassette, in that the env gene inserted into ALVAC-FL(dl IS) was deleted of the region encoding the putative IS region (3) (amino acids 315 to 341 [23]). This deletion was generated by oligonucleotide mutagenesis by a modification of the Mandecki procedure (14). ALVAC and recombinant vaccinia viruses were generated by standard procedures (20). *
Corresponding author. 2370
NOTES
VOL. 67, 1993
recombinant demonstrated that the experimental vaccine was well tolerated by the recipients and elicited protective levels of rabies virus-neutralizing antibodies (2, 8a). In order to appreciate the contribution of highly attenuated, host range-restricted avipoxvirus-based vectors for immunization against other pathogens, particularly retroviruses, we targeted the oncornavirus feline leukemia virus (FeLV). ALVAC-based FeLV vaccine candidates that contained FeLV env and gag components were engineered. ALVAC-FL (vCP97), containing the entire FeLV subgroup A (FeLV-A) Glasgow-1 env and gag genes and a portion of the pol gene (1,272 bp) sufficient to encode the 14-kDa protease, was constructed. Since the FeLV envelope glycoprotein contains a putative immunosuppressive (IS) region (3) (amino acids 315 to 341 according to the numbering of Stewart et al. [23]), a second ALVAC recombinant [ALVAC-FL(dl IS); vCP93] was derived to determine the effects of this region on FeLV env synthesis and processing and protective immune responses. ALVAC-FL(dl IS) is identical to ALVAC-FL except that the FeLV-A env gene was deleted of sequences encoding the putative IS region. A schematic representation of the FeLV-A sequences included in the ALVAC-FeLV recombinant viruses is depicted in Fig. 1. ALVAC-FL-directed env gene expression resulted in the synthesis of the precursor polypeptide of 85 kDa and proteolytically processed mature env polypeptides of 70 and 18 kDa (Fig. 2A, lane 3). This is consistent with the expression of the mature 70-kDa extracellular and 15-kDa transmembrane envelope glycoprotein domains observed in FeLVinfected cells. The reason for the apparently higher molecular mass of the transmembrane domain in ALVAC-FLinfected cells compared with that in FeLV-infected cells is not presently known. Also consistent with env gene expression in FeLV-infected cells, the expressed envelope glycoprotein was detected on the surface of ALVAC-FL-infected cells by immunofluorescence (data not shown). Deletion of the IS region from the FeLV-A env gene resulted in aberrant expression of the envelope glycoprotein in ALVAC-FL(dl IS)-infected cells. Immunoprecipitation from ALVAC-FL(dl IS)-infected cells with an FeLV-specific antiserum demonstrated the expression of a single envelope glycoprotein species with an apparent molecular mass of 83 kDa (Fig. 2A, lane 7). Proteolytic processing of the 83-kDa polypeptide to the mature extracellular and transmembrane protein species was not observed in cells infected with ALVAC-based recombinant viruses expressing the env gene product deleted of the IS region (Fig. 2A, lanes 6 and 7). Furthermore, deletion of the region of env encoding amino acids 315 through 341 resulted in improper trafficking of the envelope glycoprotein (Fig. 3). As is evident from the immunofluorescence assays, cells infected with ALVACbased recombinants expressing either a wild-type env form [ALVAC-Fenv(+)] or an IS- form [ALVAC-Fenv(-)] were shown to express the FeLV-specific antigen internally (Fig. 3f and d, respectively). Expression of the FeLV envelope glycoprotein was also detectable on the surface of ALVACFenv(+)-infected cells (Fig. 3e), whereas surface expression was not apparent on ALVAC-Fenv(-)-infected cells (Fig. 3c). Expression of an FeLV gag-specific precursor polypeptide with an apparent molecular mass of 63 kDa was observed in cells infected with either ALVAC-FL or ALVACFL(dl IS) (Fig. 2A, solid arrowheads in lanes 3 and 7, respectively). The gag-specific nature of this gene product was confirmed by the lack of detection of this protein species
Al 2 3 4 5 6 7
I..
_,
B
1
2
2371
3
.4
*p63 -
.!...*p63
FIG. 2. Immunoprecipitation from ALVAC-FeLV recombinant virus-infected cell lysates with FeLV-specific antiserum (A) or FeLV p27-specific monoclonal antibody (B). (A) Infected Vero cell lysates and immunoprecipitation analyses were performed as described previously (25) with a bovine anti-FeLV antiserum (Antibodies Inc., La Jolla, Calif.). Immunoprecipitation was performed on lysates of cells infected with parental ALVAC (lane 1), ALVACFenv(+) (lane 2), ALVAC-FL (lane 3), vaccinia virus-FeLV gag/PR recombinant (lane 4), parental vaccinia virus (lane 5), ALVACFenv(-) (lane 6), or ALVAC-FL(dl IS) (lane 7). ALVAC recombinant viruses ALVAC-Fenv (+) and ALVAC-Fenv (-) express only the FeLV env gene plus or minus the IS region (amino acids 314 to 341; 23), respectively. The vaccinia virus-FeLVgagIPR contains the identical gag expression cassette as ALVAC-FL and ALVAC-FL (dl IS). The migration positions of 14C-radiolabeled size standards (Bio-Rad) on the 10% polyacrylamide gel (5) are marked in the right-hand margin and correspond (top to bottom) to 200, 97, 68, 43, 29, 18, and 14 kDa. After electrophoresis, the gel was fixed and treated for fluorography with 1% sodium salicylate. Open arrowheads point to env-specific gene products. In lanes 2 and 3, the open arrowheads mark the positions of the 85-kDa wild-type envelope precursor and the mature p70 and p18 protein species. In lanes 6 and 7, the open arrowheads mark the position of the noncleaved 83-kDa envelope precursor expressed by the env gene deleted of the putative IS region. The solid arrowheads point to the 63-kDa gag-specific polypeptide in lanes 3, 4, and 7. No equivalent bands are seen in lane 2 or 6. (B) Immunoprecipitation was performed as described for panel A except that a p27-specific monoclonal antibody, generously provided by Hans Lutz (University of Zurich, Zurich, Switzerland), was used. Immunoprecipitation was performed on lysates of cells infected with parental ALVAC (lane 1), ALVAC-FL(dl IS) (lane 2), or ALVAC-FL (lane 3). The positions of the gag-specific precursor polypeptide (p63) and of the incompletely proteolytically processed forms are marked by solid circles. Lines in the right-hand margin indicate the migration positions of size standards of (from top to bottom) 200, 97, 68, 43, 29, 18, and 14 kDa.
in cells infected with ALVAC-based recombinant viruses expressing only the env gene (Fig. 2A, lanes 2 and 6), by the expression of a protein with an identical molecular mass in cells infected with a vaccinia virus-based recombinant expressing the same FeLV-Agag expression cassette (Fig. 2A, lane 4), and by the immunoprecipitation of a similar-sized
2372
J. VIROL.
NOTES
I
I
FIG. 3. Detection of expression of FeLV envelope glycoprotein in cells infected with ALVAC recombinant viruses. Internal and surface immunofluorescence assays were perforned as described previously (25, 27). In short, Vero cells were infected with either parental ALVAC (a and b), ALVAC-Fenv(-), expressing the IS- form of the FeLV-A env gene (c and d), or ALVAC-Fenv(+), expressing the wild-type FeLV-A env gene (e and f) at 10 PFU/cell. Expression of the envelope glycoprotein was detected by using bovine anti-FeLV as the prinmary antibody and fluorescein isothiocyanate-conjugated goat anti-bovine immunoglobulin G (Sigma; catalog no. F-7509) as the secondary antibody. (a, c, and e) Immunofluorescence analysis of nonpermeabilized cells (surface); (b, d, and f) analysis of permeabilized samples (internal).
protein with a p27-specific monoclonal antibody (Fig. 2B). Immunoprecipitation analysis with the p27-specific monoclonal antibody suggested inefficient proteolytic processing of the FeLV Gag precursor. Inefficient proteolytic processing of the FeLV Gag precursor was also reported with a feline herpesvirus vector (29). This contrasts with the efficient processing of human and simian immunodeficiency virus (HIV and SIV, respectively) gag gene products expressed by poxvirus vectors (10, 22). Differences in Gag protein processing may reflect the alternative modes of regulation for protease expression by FeLV (translation readthrough [30]) and by HIV and SIV (ribosomal frameshifting [13]). Identical env- and gag-specific gene expression and processing were observed in primary chicken embryo fibroblasts, monkey kidney cells (Vero), and Crandell feline kidney cells (data not shown). The protective efficacy of the ALVAC-based FeLV recombinant viruses was assessed by exposure of cats to a homologous FeLV-A/Glasgow-1 isolate after vaccination. In this experiment, 18 cats (8 to 9 weeks of age) were divided into three groups of six and inoculated with either ALVACFL(dl IS) (cats 1 to 6) or ALVAC-FL (cats 7 to 12) or not vaccinated (cats 13 to 18). Vaccination with either ALVACFL(dl IS) or ALVAC-FL consisted of two subcutaneous
inoculations of 108 PFU at 5 and 2 weeks prior to challenge. All cats were challenged by oronasal administration of 2 x 106 FFU of the FeLV-A/Glasgow-1 isolate to simulate natural transmission (correlates with 4 50% cat-infectious doses by this route, with no systemic corticosteroid administration). Blood samples were taken from all cats at -5, -2, 0, +3, +6, +9, and +12 weeks relative to the time of challenge for evaluating p27 antigenemia, presence of infectious FeLV, and detection of FeLV antigen in blood smears by immunofluorescence. No local or systemic adverse reactions were noted in cats given the ALVAC recombinant viruses, consistent with results of previous inoculation of ALVAC in other animal species, including humans (1, 2, 24, 26, 28; unpublished results). All six nonvaccinated control animals developed persistent viremia by 3 weeks after FeLV challenge (Table 1). Viremia was assessed by the detection of p27 antigenemia, by detection of FeLV-specific antigens in blood smears, and by the isolation of infectious FeLV from plasma. ALVAC-FL(dl IS) afforded partial protection against FeLV (Table 1). Persistent viremia developed by 3 weeks postchallenge in two cats (1 and 5) vaccinated with ALVAC-FL(dl IS). Cat 4 had no detectable p27 antigen or infectious FeLV in the blood until 12 weeks postchallenge.
VOL. 67, 1993
NOTES
2373
TABLE 1. Response of cats to challenge with FeLVa Vaccine
Cat no.
ALVAC-FL(dl IS)
1 2 3 4 5
6
ALVAC-FL
None
Result at wk: -5 (EV)
-2 (EV)
0 (EV)
--
--
--
------
------
-
-----
__ _ ++ -_
----
---_ ---
-- -_ --
+3 (EV)
+6 (FEV)
+9 (FEV)
+12 (FEV)
+ + +
+ + +
_
++ +++
+++ + +-
7 8 9 10 11 12
--
--
--
--
--
--
--
+-
13 14 15 16 17 18
- - - -- --
- - - -- --
- - - -
-_ +++
-++
_ ---
---
--
_
- -------
---
---
+ +
- + +
+ + + +
-
+ + + + + + + + +
+ + + + + + + + +
+ + +
+ + +
+ + + + +
--
++
-++
- -
+ +
- + +
--
++
-++
a FeLV p27 antigen in plasma (E) was determined with commercially available enzyme immunoassay plates (PetCheck; Idexx Corp.). Infectious FeLV in plasma (V) was determined by inoculation of a feline S+L- cell line, QN10, which is transformed after infection with FeLV (8). FeLV-specific antigen (F) detection was done with a rabbit anti-FeLV serum. Blood smears were fixed for 5 min in methanol at -20'C, washed in water, and dried; 25 Ml of rabbit anti-FeLV antibody was inoculated within a circle inscribed on the smear with a diamond pen. The smear was incubated for 1 h at 37°C in a humidified chamber, washed three times in phosphate-buffered saline (PBS) and once in distilled H20, and dried. Goat anti-cat immunoglobulin G-fluorescein isothiocyanate conjugate was inoculated onto the circle, and the smear was incubated for an additional 1 h. The smear was then washed as above, dried, and examined for immunofluorescence in a microscope with a UV light source. A single result, positive (+) or negative (-), is shown for each test at each time point; e.g., for cat 5 at week +6, FeLV-specific antigen (F) was not detected (-), p27 antigen in plasma (E) was detected (+), and infectious FeLV in plasma (V) was detected (+).
At 3 weeks postchallenge, infectious FeLV was isolated from the blood of cat 2 in the absence of p27 antigenemia. No infectious virus, however, was detectable at 6, 9, or 12 weeks postchallenge in this animal. Further, no p27 antigenemia and no specific FeLV blood-associated antigens were detected throughout the observation period. Two cats (3 and 6) remained free of viremia through 12 weeks postchallenge (Table 1). Impressively, all six cats vaccinated with ALVAC-FL resisted challenge with FeLV (Table 1). Only cat 12 demonstrated a transient p27 antigenemia at 3 weeks postchallenge. No evidence of persistent viremia was observed with other samples from this cat. Throughout the observation period following FeLV challenge, the cats given ALVAC-FL were free of p27 antigenemia, did not demonstrate FeLV antigen in blood smears, and were free of detectable infectious FeLV (Table 1). These data clearly show that expression of the putative IS region present within the FeLV envelope in an ALVAC recombinant does not adversely affect the ability to induce protective immunity against FeLV challenge. Furthermore, the ALVAC-FL(dl IS) recombinant virus expressing an aberrant form of the envelope glycoprotein provided incomplete protective immunity. This emphasizes the critical importance of a properly expressed envelope glycoprotein in an FeLV vaccine. An intriguing observation was that protection against persistent viremia occurred in the absence of detectable neutralizing antibody at the time of challenge (Table 2). None of the susceptible cats developed detectable neutralizing antibody even after FeLV challenge. Significantly, however, although none of the vaccinated cats displayed any FeLV-specific neutralizing antibody titers prior to challenge, nine of nine cats that resisted the challenge developed
TABLE 2. FeLV neutralizing-antibody titersa Antibody titer at wk: 0 +3 +6 +9
+12
0 0 0 0 0 0
0 4 4 0 0 4
0 >32 16 0 0 >32
0 0 0 0 0 0
0 0 0 0 0 0
4 0 4 4 4 4
16 >32 8 16 8 >32
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
Vaccine
Cat no.
-5
-2
ALVAC-FL(dl IS)
1 2 3 4 5 6
0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
ALVAC-FL
7 8 9 10 11 12
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
None
13 14 15 16 17 18
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
a Neutralizing antibodies were assayed by focus reduction of FeLV-A/
Glasgow-i. Equal volumes (50 p,l) of twofold dilutions of plasma and FeLV
were mixed and incubated for 2 h at 37'C. A sample (25 li) was then removed and inoculated into one well of a 12-well cluster plate seeded 24 h previously with 4 x 104 QN10 cells in 1 ml of medium containing 4 jg of Polybrene per ml. The cells were incubated at 37°C, and the medium was replaced after 2 h and again 3 days later. Foci were counted microscopically on day 6 after inoculation. Antibody titers are expressed as the reciprocal of the dilution of plasma that reduced the focus count by 75% relative to that in a virus control incubated without plasma.
2374
NOTES
neutralizing antibody activity by 9 to 12 weeks postchallenge (Tables 1 and 2). These results suggest that protection was associated with the ability to mount a neutralizing-antibody response after challenge. However, one cannot exclude the possibility that other immune mechanisms (i.e., cytotoxic T lymphocytes) were critical in the protection observed. These results demonstrate the protective efficacy of ALVAC-FL. Together with the inherent safety properties associated with the ALVAC vector, ALVAC-FL may provide a safe and effective FeLV vaccine. This is especially relevant in light of existing concerns about the efficacy of currently available FeLV vaccines (11, 17). Interestingly, a previous study reported the apparent lack of immunogenicity in cats of a vaccinia virus-based recombinant expressing the FeLV envelope glycoprotein (9). Furthermore, this vaccinia virus-FeLV env recombinant did not protect cats against an FeLV challenge (9a). It would therefore be informative from an immunobiological standpoint to evaluate the protective efficacy of ALVAC recombinants expressing the env and gag genes individually. It will also be important to determine the effector mechanisms of immunity against FeLV, since no FeLV-neutralizing antibody was present at the time of challenge. The data reported here may have more general implications for vaccine development and particularly for understanding immunity to retrovirus pathogens. As demonstrated here, parenteral (subcutaneous) administration of an ALVAC recombinant virus effectively protected against a mucosal (oronasal) FeLV challenge. The ability of parenteral administration of other poxvirus-based recombinants to protect against disease following a mucosal challenge has also been observed with avian influenza virus (27), equine influenza virus (unpublished results), canine distemper virus (28), and pseudorabies virus (la, 21). The requirement for significant levels of neutralizing antibody at the time of retrovirus exposure may not be as critical as is generally assumed. This is suggested by the data presented here as well as those from other retrovirus vaccine studies, including FeLV (4, 18), Friend murine leukemia virus (6, 15), and equine infectious anemia virus (12). Proper cellular priming of the immune system appears to be a necessary and perhaps sufficient basis for protective immunity against retroviruses. It is clear that ALVAC-FL can prime an immune response that is recalled rapidly. ALVACbased vectors may therefore have relevance to the provision of vaccines for other retroviruses, including HIV. We thank T. Moran, S. Mercer, and J. Van der Hoeven for excellent technical assistance; J. Taylor and E. Norton for generation of recombinant viruses; D. Harbour for supervising vaccine trials; and J. Taylor and W. Cox for critical reading of the manu-
script.
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NOTES
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