Centre for Applied Microbiology and Research, Porton Down, Salisbury SP4 0JG, UK1
Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Headley Way, Oxford OX3 9DS, UK 2
Department of Medicine, University Hospital of Wales, Cardiff, UK3
Author for correspondence: Christine Bruce.Fax +44 1980 611310. e-mail christine.bruce{at}camr.org.uk
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Abstract |
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Introduction |
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We have linked a replication-deficient adenovirus type 5 E1- vector with the powerful constitutive cytomegalovirus (CMV) immediate early (IE) promoter (McGrory et al., 1988 ; Wilkinson & Akrigg, 1992
). This vector replicates only in a permissive cell line (e.g. 293) that supplies the E1 gene product in trans, but can be used to infect a wide variety of non-permissive cell types. A gene placed under the control of the IE promoter is expressed at high levels but other adenovirus proteins are not normally produced (Wilkinson & Akrigg, 1992
). When the recombinant virus is injected into an animal, the expressed protein can induce antibodies and protective immunity (Jacobs et al., 1992
). Furthermore, CTL responses have been shown to be induced by this type of vector (Fooks et al., 1995
).
We describe here the construction of a replication-deficient adenovirus expressing the envelope glycoprotein gene (env) of HIV-1 strain IIIB. Expression of HIV-1 env is a complex process requiring co-expression of the HIV-1 trans-activator gene rev, which interacts with a Rev-responsive element in the env coding sequences (Feinberg et al., 1986 ; Sodroski et al., 1986
; Rosen et al., 1988
). A bicistronic adenovirus vector was therefore constructed expressing rev and env in tandem. There is also evidence that env expression may be dependent on the presence of the tat/rev 5' splice-donor site (5SD), even though splicing does not occur at this site (Chang & Sharp, 1989
; Lu et al., 1990
). However, other studies have suggested that this splice site is not required (Natuk et al., 1992
; Emerman et al., 1989
; Nasioulas et al., 1994
). We confirm that the 5SD is required for efficient Rev-stimulated env expression in plasmids and the adenovirus vector. We also show that such recombinant adenovirus vectors can induce both humoral and cellular immune responses in mice.
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Methods |
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Construction of recombinant plasmids and adenoviruses.
Cloning of plasmid DNA fragments was performed by using standard methods (Sambrook et al., 1989 ) and checked by restriction endonuclease analysis. Plasmid DNA was extracted and purified by using Qiagen columns. The HIV-1 env gene from strain IIIB clone BH10 (Ratner et al., 1985
) was inserted into a plasmid under the control of the CMV major IE promoter and polyadenylation signal, described previously (Wilkinson & Akrigg, 1992
). Specific deletions were made by restriction endonuclease digestion and ligation. The sequence surrounding the point of insertion of the oligonucleotide containing the synthetic 5SD site was determined by DNA sequencing. Adenovirus recombinants were produced according to the method of McGrory et al. (1988)
, also described previously (Wilkinson & Akrigg, 1992
). The CMV promoterenv expression cassette was inserted into the HindIII site of the adenovirus transfer vector pMV60. The resulting plasmid was co-transfected into 293 cells along with pJM17, a plasmid containing the complete adenovirus genome plus an additional prokaryotic DNA fragment inserted into the E1a gene (the adenovirus E1a gene product is provided in trans by the 293 cells). The additional fragment makes pJM17 too large to package into adenovirus capsids. Recombination between the adenovirus early sequences flanking the expression cassette in pMV60 and the complete adenovirus genome in pJM17 resulted in generation of recombinant adenovirus containing env under the control of the CMV IE promoter and polyadenylation signal. Adenovirusenv recombinants were confirmed by PCR using a 5' oligonucleotide primer in env (nt 72257245) and a 3' oligonucleotide primer adjacent to the CMV polyadenylation signal sequence, as well as primers to detect adenovirus sequences (Zhang et al., 1993
).
Transfections.
Plasmid DNA was transfected into 293 cells (5x106 cells per 25 cm2 flask) by using Transfectam (Promega) according to the manufacturer's instructions. The transfection mixture was applied to the cells in serum-free DMEM for 6 h at 37 °C and then 5 ml fresh DMEM plus FCS was added. Samples contained 5 µg DNA, comprising 3·5 µg test plasmid, 1·5 µg pMV19 (rev expression vector) (or pUC19 control) and 1 µg pIEP-gal (containing a ß-galactosidase gene as an internal control for variations in transfection efficiency) (Wilkinson & Akrigg, 1992 ). pMV19 was constructed by removing the intron from a complete rev gene (HIV-1 IIIB strain) by using a Muta-Gene in vitro mutagenesis kit (Bio-Rad) and placing it under the control of the IE promoter of CMV. rev expression by this construct was confirmed by immunofluorescence (unpublished results) and biological activity was also demonstrated by enhanced env gene expression in co-transfection experiments. After incubation for a further 2 days at 37 °C, the cells were washed with PBS and harvested by scraping into PBS, centrifuged and lysed by resuspending in 120 µl PBS containing 10% Triton X-100 (BDH). Lysates were centrifuged (Eppendorf) to remove particulate matter and 100 µl of each supernatant was assayed for Env gp160/120 by ELISA (see below) and 20 µl was assayed for ß-galactosidase activity using o-nitrophenyl galactoside (Sigma) as described previously (Wilkinson & Akrigg, 1992
).
Adenovirus infections.
Recombinant adenoviruses were plaque-purified three times by growth in 293 cells. To measure env gene expression, titrated virus was applied to MRC5 cells (1x106 cells per 6 cm Petri dish) in 1·5 ml DMEM containing 10% FCS. Adenovirus env recombinants were applied at an m.o.i. of 30 and RAd46 ( rev recombinant) at an m.o.i. of 10. After incubation for 4 h at 37 °C, 5 ml DMEM containing 10% FCS was added and incubation was continued for 3 days at 37 °C. Cells were then washed with PBS and lysed with Triton X-100 as described above. Triton X-100 was used to lyse the cells, releasing cellular proteins including HIV-1 Env synthesized by the recombinant adenovirus, but does not affect the virus.
Larger-scale virus preparations were made for electrophoresis and blotting. 3·5x107 293 cells in a 175 cm2 flask were incubated with each recombinant adenovirus at an m.o.i. of 1 until cells were released from the flask surface. Infected cells were harvested by centrifugation and lysed with 1·5 ml Triton X-100 in PBS.
To produce virus for inoculation of mice, ten 175 cm 2 flasks of 293 cells were incubated with each recombinant adenovirus at an m.o.i. of 1 until extensive CPE was seen and cells had been released from the flask surface. The supernatant was cleared of cellular debris by centrifugation (10 min, 12000 g) and a virus pellet was obtained by centrifugation (25000 r.p.m. for 2·5 h in a Sorval AH 629 rotor). The virus pellet was resuspended in PBS and a small aliquot was used in a plaque assay to determine the virus titre.
ELISA for env gene expression.
The ELISAs were performed essentially as described by Jones et al. (1995) . Each well of a 96 well plate (Nunc maxisorb) was coated with 50 µl 1 µg/ml Galanthus nivalis lectin (Sigma) in PBS at 4 °C overnight. After washing with PBS containing 0·05% Tween 20 (Sigma), wells were blocked with 100 µl PBS containing 5% dried milk (Marvel) and 0·05% Tween for 1 h at 37 °C. Plates were washed as before and a twofold dilution series of 50 µl test samples diluted in PBS was applied across the plate. After incubation for 2 h at 37 °C and washing, 50 µl of a 1:100 dilution of rabbit anti-HIV gp120 in blocking buffer was applied to each well and the plates were incubated for 1 h at 37 °C. Plates were washed and bound antibody was detected with 50 µl of a 1:5000 dilution of affinity-purified, peroxidase-conjugated donkey anti-rabbit immunoglobulin (Jackson Immuno-Research Labs) in blocking buffer. After incubation for 1 h at 37 °C, the plates were washed. Bound peroxidase was detected with 50 µl TM Blue substrate (Sigma), the plates were incubated for 15 min at 20 °C and the reaction was stopped by adding 50 µl 1 M H2SO4. The concentration of bound gp120 was estimated by comparing the A450 with standard samples of known gp120 concentration (a generous gift from D. Jones, CAMR, Porton Down, UK).
SDSPAGE and Western blotting.
Triton X-100 lysates of 293 cells infected with recombinant adenoviruses were clarified by centrifugation (Eppendorf) for 20 min. Supernatants were subjected to SDSPAGE and electroblotted onto nitrocellulose filters (Amersham) as described by Polyanskaya et al. (1997) . Blots were blocked with 10% FCS in PBS containing 0·05% Tween 20 and probed with the same anti-gp120 and peroxidase-linked antibodies used for the ELISA. Proteins were visualized by using enhanced chemiluminescence (ECL, Amersham).
Animals and administration of viruses.
BALB/c mice (46 weeks old) were inoculated intraperitoneally (i.p.) with either 5x107 or 1x108 p.f.u. of the appropriate adenoviruses in 500 µl PBS. RAd142, RAd501 or RAd501 plus RAd46 were used. Mice were inoculated at the times detailed in each experiment.
ELISA for anti-gp120 antibody production.
Assays were performed in flat-bottomed microtitre plates (Nunc maxisorb). Plates were coated with 50 µl of either purified HIV- 1 GB8 gp120 (a generous gift from D. Jones, CAMR) or purified, baculovirus-produced recombinant HIV-1 IIIB gp120 (obtained from the ARP/programme EVA, NIBSC, UK). The antigen was diluted in bicarbonate coating buffer to 1 µg/ml and incubated overnight at 37 °C. Plates were washed with wash buffer (PBS containing 0·05% Tween) and wells were blocked with 60 µl wash buffer containing 5% dried milk powder (Marvel) for 1 h at 37 °C. Plasma samples were titrated as twofold serial dilutions across the plate and the antibody was allowed to bind for 2 h at 37 °C. The plate was washed five times before adding 50 µl of a 1:500 dilution of horseradish peroxidase- conjugated protein G. After 30 min incubation, the plate was washed and the bound peroxidase was detected with 50 µl TM Blue substrate (Sigma), the plates were incubated for 15 min at 20 °C and the reaction was stopped by adding 50 µl 1 M H2SO4. Antibody levels were estimated by measuring the A450. All assays included a positive antiserum produced in rabbit as a control.
CTL assays
In vitro expansion of precursor (p) CTL.
HIV-1 IIIB Env glycoprotein contains an H-2Dd-restricted CTL epitope, RGPGRAFVTI (Takahashi et al., 1993 ). The corresponding peptide was used to restimulate immune splenocytes in vitro as described previously (Hanke et al., 1998 a
, b
). Briefly, splenocytes from individual mice were isolated 14 or 28 days, respectively, after immunization with 108 or 5x107 p.f.u. and separately incubated in 10 ml lymphocyte medium (RPMI 1640 supplemented with 10% FCS, penicillin/streptomycin 100 U/ml, 20 mM HEPES and 15 mM ß-mercaptoethanol) in the presence of 2 µg/ml peptide for 5 days.
Target cells and 51Cr-release assay.
On the day of assay, effector cells were washed three times, suspended in 10% FCS RPMI medium and, in duplicates, diluted twofold in U-bottomed wells (96 well plate; Costar) to give effector to target ratios of 100:1, 50:1 and 25:1. 51Cr-labelled P815 target cells (5000 cells) were added to the effector cells in medium containing 0·1 µM peptide and incubated at 37 °C for 5 h. Effector cells were also combined with targets in the absence of peptide to determine the percentage of no-peptide background lysis. Spontaneous and total 51Cr release were estimated from wells in which target cells were incubated in medium alone or with 5% Triton X-100. The percentage specific lysis was determined as 100x(sample release-spontaneous release)/(total release-spontaneous release). To calculate the net specific release, no-peptide lysis was subtracted from the peptide- specific lysis. The spontaneous release was less than 10% of the total release.
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Results |
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To test the ability of the 5SD alone to stimulate Rev-dependent env expression, a short oligonucleotide including the 5SD was synthesized and cloned adjacent to the SspI site (nt 5736) in IEP-env to give IEP-oligo-env. The presence of the 5SD sequence restored gp120 expression completely to a level exceeding that of the bicistronic construct IEP-rev-env (Fig. 1).
(ii) Expression from recombinant adenoviruses. To determine whether the 5SD would exert a similar effect on env expression in the context of an adenovirus genome, recombinant adenoviruses Adenv-R, RAd501 and RAd142 were constructed by using the IEP-env, IEP-5SD-env and IEP-rev-env expression cassettes, respectively. When MRC5 cells were infected with each of the recombinant adenoviruses (Fig. 2 a), if necessary co-infecting with RAd46 (rev) to supply rev in trans, only RAd501 and RAd142 (revenv) produced gp120 efficiently. Therefore, the 5SD also appeared to be required for env expression in a different background (adenovirus) and in a non-immortalized cell line (MRC5).
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Immunization with recombinant adenoviruses induces humoral immune responses
We first sought to determine whether RAd142 produced a humoral response after i.p. inoculation at 0 and 3 weeks of either a high dose (1x108 p.f.u.) or a low dose (5x107 p.f.u.) of virus. Antibody levels were measured at 2, 4 and 6 weeks. As shown in Fig. 3, 3/3 animals receiving the high dose of virus produced antibodies by week 4. Two of three animals receiving the low dose of virus had also responded, but produced lower titres.
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Further experiments using the high dose of virus were carried out, again using three groups of animals injected with RAd142, RAd501 or RAd501+RAd46. Animals were inoculated i.p. with 1x108 p.f.u. of each virus, either twice at weeks 0 and 3 with antibody levels being estimated at weeks 2, 4 and 5, or only once with antibodies being measured at week 2. Again, the RAd142 group of animals produced antibodies by week 4 (after two inoculations), giving a mean log10 end-point titre of 2·51, but not at week 2 (after one inoculation). No significant antibody production was seen in either of the other two groups. Animals were also assayed for CTL responses.
Immunization with recombinant adenoviruses induces CTL responses
(i) Mice immunized three times with 5x107 p.f.u. virus. Splenocytes from four mice immunized with RAd142 (rev env) had HIV Env-specific CTL activity, as did splenocytes from 4/5 mice immunized with RAd501 (env) +RAd46 ( rev), although the responses were weaker in two of these animals than those seen in RAd142-immunized animals. One mouse immunized with RAd501 alone had a weak response. The mean activity for each group of responders is shown in Fig. 4. One mouse in each of the RAd142 and RAd501 groups died, for reasons unknown, before the end of this experiment, but it was thought unlikely to be related to the immunization.
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Discussion |
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The presence of the 5SD sequence was also required for efficient expression of the env gene in the context of an adenovirus vector and in different cell types, although it was possible to obtain detectable levels of env expression without the 5SD site if MRC5 cells were infected with Adenv-R plus RAd46 at high m.o.i. for at least 6 days (data not shown). The mechanism by which the 5SD sequence regulates env gene expression is not understood, but seems to be linked either directly or indirectly to the mechanism of Rev trans-activation (Chang & Sharp, 1989 ; Lu et al. , 1990
; Hammarskjold et al., 1994
; Kjems & Sharp, 1993
; Barksdale & Baker, 1995
; Mikaelian et al., 1996
). We have shown that significant levels of HIV-1 gp120 can be produced by recombinant adenoviruses either by expressing rev bicistronically (RAd142) or by providing rev in trans on a separate adenovirus (RAd501 plus RAd46) when grown in vitro.
We next used these constructs in vivo to investigate their immunogenicity. It has been shown previously that a replication- deficient adenovirus expressing the lacZ gene can induce a long-term humoral and cellular response after a single inoculation (Juillard et al., 1995 ) and that protective immune responses can be obtained (Jacobs et al., 1992
; Fooks et al., 1998
) with the administration of some recombinant adenoviruses. It is clear from our studies that the bicistronic virus, RAd142, is immunogenic and can produce both humoral and CTL responses in mice. The humoral response required two inoculations and was dose dependent, requiring 1x108 p.f.u. to give a consistent response. Providing rev in trans was thought less likely to give a good immunogenic response, as this requires the simultaneous infection of any given cell by the two viruses. Others (Chenciner et al., 1997
) have shown, however, that simultaneous infection with three recombinant adenoviruses, expressing the simian immunodeficiency virus (SIV) Env protein and the HIV-1 Tat and Rev proteins, can be successful in inducing anti-SIV Env antibodies. Dual infection with our two recombinant adenoviruses has not been found to be successful in producing an antibody response in any experiment carried out. We, however, used a lower level of each virus (1x108 p.f.u.) compared with that used by Chenciner et al. (1997)
(1x109 p.f.u.), and this could have resulted in a smaller number of cells being doubly infected and thus a lower level of Env being produced. Simultaneous infection with our two adenoviruses did induce a CTL response, however, even when low doses of virus were used. A CTL response could also be detected after a single inoculation of adenovirus, whereas an antibody response always required two inoculations. This suggests that only a very low level of expression is required for the adenovirus to produce a CTL response compared with that required for a humoral response. The fact that RAd501 on its own could elicit a CTL response, albeit weak, supports the idea that only low-level expression of env is required. CTL responses in mice in the absence of serum IgG have also been described after mucosal administration of recombinant adenovirus expressing SIV p55 Gag from the E3 region (Flanagan et al., 1997
). Taken together, these data suggest that it may be possible to manipulate the immune response to the transgene product by altering the dose of virus administered, the number of immunizations and the route of administration. Low doses of virus or single inoculations can produce a cellular response, whereas high levels of virus are required to elicit a humoral response as well. It should be noted, however, that the CTL responses were detected after expansion in vitro of pCTL, whereas humoral responses were analysed by detection of secreted product, i.e. antibody. Further work will reveal whether low doses of recombinant adenovirus can prime B cells in the absence of antibody production. It is possible that good humoral responses could be obtained by using a combination vaccination strategy, such as priming with a recombinant adenovirus followed by an antigenic boost with an alternative vector or protein.
We were interested to observe that a CTL response was induced by using the trans-activated env construct. It has proved technically difficult to produce bicistronic adenovirus constructs, although the monocistronic versions are relatively easy to construct. We have constructed several other monocistronic env- recombinant adenoviruses that have been made by using field isolates from Uganda (representing clades A and D) and the clinical isolate W61D (clade B). Studies using these constructs expressing more relevant HIV- 1 isolates will now be possible, including analysis of protective efficacy as cognate HIV-1 envSIV chimeras (SHIVs) become available.
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Acknowledgments |
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References |
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Barksdale, S. K. & Baker, C. C. (1995). The human immunodeficiency virus type 1 Rev protein and the Rev-responsive element counteract the effect of an inhibitory 5' splice site in a 3' untranslated region. Molecular and Cellular Biology 15, 2962-2971 .[Abstract]
Chanda, P. K. , Natuk, R. J. , Mason, B. B. , Bhat, B. M. , Greenberg, L. , Dheer, S. K. , Molnar-Kimber, K. L. , Mizutani, S. , Lubeck, M. D. , Davis, A. R. & Hung, P. P. (1990). High level expression of the envelope glycoproteins of the human immunodeficiency virus type I in presence of rev gene using helper-independent adenovirus type 7 recombinants. Virology 175, 535-547.[Medline]
Chang, D. D. & Sharp, P. A. (1989). Regulation by HIV Rev depends upon recognition of splice sites. Cell 59, 789-795.[Medline]
Chenciner, N. , Randrianarison-Jewtoukoff, V. , Delpeyroux, F. , Hanania, N. , Pedroza Martins, L. , Stratford Perricaudet, L. , Perricaudet, M. & Wain-Hobson, S. (1997). Enhancement of humoral immunity to SIVenv following simultaneous inoculation of mice by three recombinant adenoviruses encoding SIVenv/poliovirus chimeras, Tat and Rev. AIDS Research and Human Retroviruses 13, 801-806.[Medline]
Dewar, R. L. , Natarajan, V. , Vasudevachari, M. B. & Salzman, N. P. (1989). Synthesis and processing of human immunodeficiency virus type 1 envelope proteins encoded by a recombinant human adenovirus. Journal of Virology 63, 129-136.[Medline]
Emerman, M. , Vazeux, R. & Peden, K. (1989). The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization. Cell 57, 1155-1165 .[Medline]
Feinberg, M. B. , Jarrett, R. F. , Aldovini, A. , Gallo, R. C. & Wong-Staal, F. (1986). HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell 46, 807-817.[Medline]
Flanagan, B. , Pringle, C. R. & Leppard, K. N. (1997). A recombinant human adenovirus expressing the simian immunodeficiency virus Gag antigen can induce long-lived immune responses in mice. Journal of General Virology 78, 991-997.[Abstract]
Fooks, A. R. , Schadeck, E. , Liebert, U. G. , Dowsett, A. B. , Rima, B. K. , Steward, M. , Stephenson, J. R. & Wilkinson, G. W. G. (1995). High-level expression of the measles virus nucleocapsid protein by using a replication-deficient adenovirus vector: induction of an MHC-1- restricted CTL response and protection in a murine model. Virology 210, 456-465.[Medline]
Fooks, A. R. , Jeevarajah, D. , Lee, J. , Warnes, A. , Niewiesk, S. , ter Meulen, V. , Stephenson, J. R. & Clegg, J. C. S. (1998). Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins: protective immune responses in rodents. Journal of General Virology 79, 1027-1031 .[Abstract]
Graham, F. L. , Smiley, J. , Russell, W. C. & Nairn, R. (1977). Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Journal of General Virology 36, 59-74.[Abstract]
Hammarskjold, M.-L. , Li, H. , Rekosh, D. & Prasad, S. (1994). Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron. Journal of Virology 68, 951-958.[Abstract]
Hanke, T. , Schneider, J. , Gilbert, S. C. , Hill, A. V. S. & McMichael, A. (1998a). DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum: immunogenicity in mice. Vaccine 16, 426 -435.[Medline]
Hanke, T. , Blanchard, T. J. , Schneider, J. , Hannan, C. M. , Becker, M. , Gilbert, S. C. , Hill, A. V. S. , Smith, G. L. & McMichael, A. (1998b). Enhancement of MHC class I- restricted peptide-specific T cell induction by a DNA prime/MVA boost vaccination regime. Vaccine 16, 439 -445.[Medline]
Imler, J.-L. (1995). Adenovirus vectors as recombinant viral vaccines. Vaccine 13, 1143-1151 .[Medline]
Jacobs, S. C. , Stephenson, J. R. & Wilkinson, G. W. G. (1992). High- level expression of the tick-borne encephalitis virus NS1 protein by using an adenovirus-based vector: protection elicited in a murine model. Journal of Virology 66, 2086-2095 .[Abstract]
Jones, D. H. , McBride, B. W. , Roff, M. A. & Farrar, G. H. (1995). Efficient purification and rigorous characterisation of a recombinant gp120 for HIV vaccine studies. Vaccine 13, 991-999.[Medline]
Juillard, V. , Villefroy, P. , Godfrin, D. , Pavirani, A. M. , Venet, A. & Guillet, J.-G. (1995). Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector. European Journal of Immunology 25, 3467-3473 .[Medline]
Kjems, J. & Sharp, P. A. (1993). The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly. Journal of Virology 67, 4769-4776 .[Abstract]
Lu, X. B. , Heimer, J. , Rekosh, D. & Hammarskjold, M.-L. (1990). U1 small nuclear RNA plays a direct role in the formation of a rev-regulated human immunodeficiency virus env mRNA that remains unspliced. Proceedings of the National Academy of Sciences, USA 87, 7598-7602 .[Abstract]
Lubeck, M. D. , Natuk, R. J. , Chengalvala, M. , Chanda, P. K. , Murthy, K. K. , Murthy, S. , Mizutani, S. , Lee, S.-G. , Wade, M. S. , Bhat, B. M. , Bhat, R. , Dheer, S. K. , Eichberg, J. W. , Davis, A. R. & Hung, P. P. (1994). Immunogenicity of recombinant adenovirushuman immunodeficiency virus vaccines in chimpanzees following intranasal administration. AIDS Research and Human Retroviruses 10, 1443-1449 .[Medline]
McGrory, W. J. , Bautista, D. S. & Graham, F. L. (1988). A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology 163, 614-617.[Medline]
Mikaelian, I. , Krieg, M. , Gait, M. J. & Karn, J. (1996). Interactions of INS (CRS) elements and the splicing machinery regulate the production of Rev-responsive mRNAs. Journal of Molecular Biology 257, 246-264.[Medline]
Nasioulas, G. , Zolotukhin, A. S. , Tabernero, C. , Solomin, L. , Cunningham, C. P. , Pavlakis, G. N. & Felber, B. K. (1994). Elements distinct from human immunodeficiency virus type 1 splice sites are responsible for the Rev dependence of env mRNA. Journal of Virology 68, 2986-2993 .[Abstract]
Natuk, R. J. , Chanda, P. K. , Lubeck, M. D. , Davis, A. R. , Wilhelm, J. , Hjorth, R. , Wade, M. S. , Bhat, B. M. , Mizutani, S. , Lee, S. , Eichberg, J. , Gallo, R. C. , Hung, P. P. & Robert-Guroff, M. (1992). Adenovirushuman immunodeficiency virus (HIV) envelope recombinant vaccines elicit high- titered HIV-neutralizing antibodies in the dog model. Proceedings of the National Academy of Sciences, USA 89, 7777-7781 .[Abstract]
Natuk, R. J. , Lubeck, M. D. , Chanda, P. K. , Chengalvala, M. , Wade, M. S. , Murthy, S. C. S. , Wilhelm, J. , Vernon, S. K. , Dheer, S. K. , Mizutani, S. , Lee, S.-G. , Murthy, K. K. , Eichberg, J. G. , Davis, A. R. & King, P. P. (1993). Immunogenicity of recombinant human adenovirushuman immunodeficiency virus vaccines in chimpanzees. AIDS Research and Human Retroviruses 9, 395-404.[Medline]
Polyanskaya, N. , Sharpe, S. , Cook, N. , Leech, S. , Banks, J. , Dennis, M. , Hall, G. , Stott, J. & Cranage, M. P. (1997). Anti-major histocompatibility complex antibody responses to simian B cells do not protect macaques against SIVmac infection. AIDS Research and Human Retroviruses 13, 923-931.[Medline]
Ratner, L. , Haseltine, W. , Patarca, R. , Livak, K. J. , Starcich, B. , Josephs, S. J. , Doran, E. R. , Rafalski, J. A. , Whitehorn, E. A. , Baumeister, K. , Ivanoff, L. , Petteway, S. R.Jr , Pearson, M. L. , Lautenberger, J. A. , Papas, T. S. , Ghrayeb, J. , Chang, N. T. , Gallo, R. C. & Wong-Staal, F. (1985). Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277-284.[Medline]
Rosen, C. A. , Terwilliger, E. , Dayton, A. I. , Sodroski, J. G. & Haseltine, W. A. (1988). Intragenic cis -acting art gene-responsive sequences of the human immunodeficiency virus. Proceedings of the National Academy of Sciences, USA 85, 2071-2075 .[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sodroski, J. , Goh, W. C. , Rosen, C. A. , Dayton, A. , Terwilliger, E. & Haseltine, W. A. (1986). A second post- transcriptional trans-activator gene required for HTLV-III replication. Nature 321, 412-417.[Medline]
Takahashi, H. , Nakagawa, Y. , Yokomuro, K. & Berzofsky, J. A. (1993). Induction of CD8 + cytotoxic T lymphocytes by immunization with syngeneic irradiated HIV-1 envelope derived peptide-pulsed dendritic cells. International Immunology 5, 849-857.[Abstract]
Van Beveren, C. , Coffin, J. & Hughes, S. (1985). HTLV-3 genome. In RNA Tumor Viruses, 2nd edn, Supplements and Appendices, pp. 1109-1123. Edited by R. Weiss, N. Teich & J. Coffin. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Wilkinson, G. W. G. (1994). Gene therapy and viral vaccination. Reviews in Medical Microbiology 5, 97-106.
Wilkinson, G. W. G. & Akrigg, A. (1992). Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector. Nucleic Acids Research 20, 2233-2239 .[Abstract]
Zhang, W. W. , Fang, X. , Branch, C. D. , Mazur, W. , French, B. A. & Roth, J. A. (1993). Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis. Biotechniques 15, 868-872.[Medline]
Received 31 March 1999;
accepted 8 June 1999.