Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV
© Pang et al; licensee BioMed Central Ltd. 2001
Received: 24 September 2001
Accepted: 13 November 2001
Published: 13 November 2001
Toward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons.
We cloned into a Δpre-M/E dengue virus replicon genes for either green fluorescent protein (GFP), HIV gp160 or HIV gp120 and tested the ability of these constructs to express dengue virus proteins as well as the heterologous proteins in tissue culture after transfection of replicon RNA.
Heterologous proteins were readily expressed from these constructs. GFP and gp120 demonstrated minimal or no toxicity. Gp160 expressing replicons were found to express proteins abundantly at 36 hours post transfection, but after 50 hrs of transfection, few replicon positive cells could be found despite the presence of cellular debris positive for replicon proteins. This suggested that gp160 expressed from dengue virus replicons is considerably more toxic than either GFP or gp120. The successful expression of heterologous proteins, including HIV gp120 for long periods in culture suggests this vector system may be useful as a vaccine vector, given appropriate delivery methods.
Despite tremendous progress in developing anti-retroviral drugs to combat HIV, there remains a need for an effective HIV vaccine. This need is particularly pressing in third world countries, where demographics and economics make drug therapy difficult to deliver. Although HIV infection elicits neutralizing antibodies and a cellular immune response against the virus [reviewed in  &] and there exist "exposed uninfected" (EU) individuals that appear to have acquired resistance to infection by HIV [3, 4], the hallmark of HIV infection is the almost universal inability of humans to mount an immune response that can prevent the eventual development of AIDS.
An effective vaccine will require not only the design of effective immunogens, but also the design of optimized protocols of immunogen delivery. As a live, attenuated vaccine for HIV is considered difficult to test and dangerous to implement [1, 2, 5–9], various alternatives to HIV could be considered as potential "live" vectors for HIV immunogens, including enteric bacteria, poxviruses (vaccinia and canarypox), small RNA viruses (e.g. poliovirus and Semliki Forest virus), Rhabdoviruses (e.g. vesicular stomatitis virus), DNA viruses (e.g. adenovirus and adeno-associated viruses) and even naked DNA to achieve expression in living host cells [2, 10].
Dengue possesses several advantages which favor its choice as a vector for HIV immunogens. As a flavivirus, it replicates entirely in the cytoplasm through RNA directed RNA polymerization and is incapable of integrating into the host genome. Flavivirus replicons can replicate inside cells and achieve prolonged expression of high levels of virally encoded proteins with minimal toxicity [11, 12] and are unable to recombine or mutate to produce infectious HIV particles. Finally, by eliciting an immune reaction against the dengue non-structural proteins remaining in replicons, dengue virus replicons may induce a protective immunity against dengue which would not predispose vaccinated individuals to DHF. Properly administered, dengue virus replicons expressing HIV epitopes might thus serve as dual vaccines, conferring protection against dengue virus as well as HIV.
The challenges in developing a safe and effective HIV vaccine are many and varied. Choice of immunogen is clearly problematic. Critical epitopes may be masked by glycosyl groups and/or tertiary structure [13–15] and  The extensive genetic variability of HIV complicates immunogen choice and the high rate of mutation increases the likelihood of the rapid development of resistance. Furthermore, the method of immunogen delivery (e.g. purified subunits or inactivated virus vs. various forms of "live" expression) can determine the relative nature and extent of humoral and cell mediated immunologic responses. Priming with various types of "live" expression followed by boosting with purified subunits is currently favored as a method to obtain stronger immunologic responses that either method alone [reviewed in  and ]. Previously acquired immunity to a viral vector such as vaccinia may influence its efficacy in inducing immunity against heterologous proteins being delivered [16–19] and it may be wise to provide physicians with HIV vaccines based on a variety of vectors to handle a variety of clinical situations.
The use of live dengue as a vaccine or as a vector for heterologous immunogens has historically been considered problematic because of the pathologies associated with dengue infection. Although dengue fever (DF) is usually self limited, dengue hemorrhagic fever (DHF) is considerably debilitating and frequently fatal . However, DHF is unlikely to result from or be promoted by the vectors reported here. The enhanced replication of virus seen in dengue hemorrhagic fever is generally seen upon reinfection by dengue virus of a serotype different from previous infections and is believed to be mediated by antibodies against viral structural proteins: so called antibody dependent enhancement of infection, or ADE. These cross reacting antibodies actually promote viral uptake by macrophages [21, 22]. The main challenge in using live dengue in humans is thus avoiding the development of antibody dependent enhancement(ADE) of infection by antibodies against the pre-M and E proteins of one dengue strain which weakly cross react with the pre-M and E of a second infecting dengue strain. Since the replicons reported here lack the major viral structural protein genes, they are not only incapable of sustaining a spreading infection but also are incapable of eliciting antibodies against the missing structural proteins. They should neither induce DF nor promote DHF.
Two strategies suggest themselves for circumventing the problem of ADE from dengue vaccination. One strategy is to immunize with multiple strains of dengue virus to elicit high affinity, neutralizing antibodies against the multiple dengue serotypes. At least one vaccine to do this (using dengue vaccine candidates, DEN-1 PDK13, DEN-2 PDK53, DEN-3 PGMK 30/F3, and DEN-4 PDK48) has been in clinical trials [27, 28]. A second strategy is to induce immunity only to viral proteins other than pre-M and E. Several studies have shown that the nonstructural glycoprotein NS1 can play an important role in protection against dengue. Mice immunized with purified dengue-2 NS1 protein injected intramuscularly and boosted after 3 days and two weeks were protected from developing lethal dengue encephalitis upon subsequent challenge with dengue 2 virus. . Similarly, mice immunized with recombinant vaccinia virus expressing authentic NS1  were protected against the development of dengue-4 virus encephalitis when challenged by intracerebral injection. Inoculation of mice with specific combinations of monoclonal antibodies (Mabs) directed against dengue-2 NS1  also protects against lethal virus encephalitis upon intracerebral dengue-2 challenge. Other nonstructural proteins are also immunogenic and may participate in eliciting protection .
Previously we have reported the successful construction of several dengue virus replicons which replicate intracellularly without the pre-M and E proteins required to form infectious virions, including one replicon which can be expressed from transfected DNA . Towards the goal of devising a "live" dual vaccine based on only non-structural dengue proteins and heterologous HIV material, we report here that these replicons can be harnessed to express heterologous genes, including HIV gp160 and gp120. Upon introduction into a host's cells, these sub-genomic fragments should replicate intracellularly and support prolonged expression of dengue and heterologous immunogens without producing the deleted dengue structural proteins and without forming infectious virions.
Our finding that dengue virus replicons can express heterologous genes, including HIV envelop, for prolonged periods of time in cell culture without selection represents a significant step in developing a new vector system potentially capable of delivering immunogens to any host in whose cells the dengue replicons can replicate. Flavivirus replicons have previously been demonstrated to express heterologous genes for up to 41 days in tissue culture in Kunjin . However, these experiments were done in the presence of selection for the heterologous genes cloned into the replicons. We have demonstrated heterologous gene expression in the absence of selection for up to at least 9 days post transfection with chimeric dengue replicons. Although we have formally demonstrated expression, not replication, our previous demonstration of the replication  of dengue replicons lacking heterologous material suggests that the replicons described here, which contain heterologous material, are indeed replicating. Evidence that cells continue to replicate and express replicon proteins in both daughter cells after transfection with these chimeric replicons further supports the implication of chimeric replicon replication. Ideally, to serve as dual vaccines against dengue as well as against other pathogens, the replicons should express the dengue NS1 protein [29–32]. So far, attempts to visualize NS1 production by Western blots have failed, presumably because of the low transfection efficiencies. However, we have previously argued that the replication of dengue replicons could not take place in the absence of the essential non-structural gene, NS1, which implies that NS1 is being made. The frequencies and fluorescence intensities of replicon positive cells seen in the experiments reported here are comparable to those seen for dengue replicons lacking heterologous material , suggesting that replication of the Δpre-M/E replicons containing heterologous material is occurring as well. The finding of at least one closely apposed pair of cells expressing high levels of replicon proteins on day 9 post transfection, two days after trypsinization and replating (Figure 7, right panel), not only implies replicon replication, but also implies the expression of NS-1 protein as well. The definitive demonstration of effective NS-1 production will have to await studies of the immune response in animals immunized with these replicons.
Choice of immunogen remains problematic for these vectors. Clearly, HIV-1 gp160 is too toxic for prolonged expression. Even the gp120-expressing replicon seems mildly toxic in that the frequency of gp120 positive cells declines with time in culture post transfection with Δpre-M/E-gp120 (not shown). However, as noted above, we have seen at least one instance of putative cellular division at least 7 days after being successfully transfected by Δpre-M/E-gp120 (Figure 7, right panel). Experiments are now in progress to determine the feasibility of long term expression of other HIV-1 immunogens, including gag and tat.
Demonstration of long term protein expression by a gp120-expressing replicon alone, of course, does not demonstrate that the chimeric dengue replicons constitute an effective vaccine. However, at the very least they add to the potential armamentarium available to the vaccinologist. It is highly likely that a successful HIV vaccination protocol will involve multiple immunogens and delivery protocols. For instance, mice immunized with attenuated Friend leukemia virus (FLV) develop an immune response whose efficacy is dependent on the additive effects of at least three separable spleen cell populations . By analogy, it may be necessary to devise multiple strategies to obtain a similarly complex and effective immune response in humans against HIV. In animal models of HIV, different immunogens and modes of immunization can induce different modes of protection with varying degrees of effectiveness [34–40] Harnessing multiple immune responses may be the answer to designing an effective HIV vaccine  and the availability of multiple vectors may facilitate the harnessing of multiple responses.
Much work obviously remains to be done to develop dengue replicons as effective vectors for HIV immunogens. More work needs to be done on determining the ability of dengue replicons to express other potential immunogens, including HIV-1 gag, tat (reviewed in 41) and env genes with deleted hypervariable regions [see ]. Animal studies in our lab are currently being initiated to determine the nature of the immune response such vectors can induce in mice and these studies will presumably need to be extended to primates. Finally, effective delivery systems will need to be devised. Although our successful development of a plasmid which can express a dengue replicon from transfected DNA facilitates delivery by DNA vaccination , the development of packaging cell lines which can package these replicons into virions would be a major step forward towards a vaccine which could be conveniently administered in typical clinical situations. Experiments are currently underway in our laboratory to develop such packaging cell lines.
Materials and methods
Culturing of dengue Virus
Dengue virus strains DEN1/WP and DEN2/NGC, kindly provided by Dr. Lewis Markoff, [43, 44] were passaged in monkey LLC-MK2 cells at 37°C in a humidified incubator under 5% CO2, using Medium 199 plus 10% fetal bovine serum (FBS) and 50 ug of Gentamicin per ml. The cells were trypsinized a day before virus infection and plated to reach approximately 80% confluence on the day of infection. Infections were typically at an MOI of 0.01 PFU/cell in Medium 199 plus 2% FBS.
In vitro mutagenesis
Heterologous genes were cloned into the previously described Δpre-M/E replicon , into the position previously occupied by the pre-M/E genes. DNA fragments used for desired regions of heterologous genes (see Figure 1) were synthesized by polymerase chain reaction (PCR) from short overlapping primers. For the Green Fluorescent Protein (GFP) gene, the 5' primer was 5'CGAAAAAAGGCGAGAAATACGCCTTTCAATATGCTGAAACGCGAGAGAATGGTGAGCAAGGGCGAGGAGCTG3' and the 3' primer was 5'AAGGTCAAAATTCAACAGCTGCTTGTACAGCTCGTCCATGCC3'. For HIV-1 gp120 gene, the 5' primer was 5'ATCATTATGCTGAATCCAACAGTGATGGCGTTCCATTTACCACACGTAACATGAGAGTGATGGGGATCAGGAAG3' and the 3' primer was 5'AAGGTCAAAATTCAACAGCTGGGTGGGTGCTAATCCTAATGGTTC3'. GFP and HIV-1 gp120 were fused with FMDV/2A self cleaving protein sequence to replace natural cleavage sites in the dengue polyprotein. These sites seem to loose their activity when juxtaposed with heterologous material (data not shown). PCR was used to amplify DNA coding for the FMDV/2A self cleaving protein The 5' primer was 5'CAGCTGTTGAATTTTGACCTTCTTAAGCTTGCGGGAGACGTCGAGTCCAACCCTGGCCCC3' and the 3' primer was 5'ATACAGCGTCACGACTCCCACCAATACTAGTGACACAGACAGTGAGGTGCTGGGGCCAGGGTTGGACTCGAC.
Dengue-2 virus cDNA cloned in the yeast shuttle vector pRS424, linearized by excision of a short Bam H1 fragment was transfected into competent  S. cerevisiae YPH857 (kindly provided by Barry Falgout, (CBER/FDA), along with the appropriate PCR fragment spanning the desired deletion. Yeast colonies which grew on tryptophan minus plates represented vectors which had recircularized by homologous recombination with these PCR fragments . DNA from these colonies was transformed into E. coli STBL 2 cells (Life Technologies, Inc.) to make sufficient quantities of dengue recombinant, genomic-length DNAs for characterization and analysis.
Expression of virus & replicons in cells
The full length virus and replicon cDNA plasmids isolated from STBL 2 cells were linearized with Sac I, purified by Qiagen chromatography, and eluted by RNAase-free water in preparation for transcription. The transcription reaction mixtures contained lug of linearized DNA; 0.5 mM(each) ATP, CTP, and UTP; 0.1 mM GTP; 0.5 mM cap analog (NEBL); 10 mM DTT; 40 U of Rnasin (Promega); 30 U of SP6 RNA polymerase; and 1 × SP6 RNA polymerase buffer (Promega) in a volume of 30 ul. The reaction mixtures were incubated at 400°C for 2 hr. Aliquots (12.5 ul) of the reaction mixtures, containing full length viral RNA, were used to transfect approximately 2 × 106 Monkey LLC-MK2 cells in phosphate-buffered saline (PBS) by electroporation in a 0.4 cm gap electroporation cuvette. Each cuvette was pulsed at 200 V, 950 uF using a BioRad Genepuls electroporator. The cells were then resuspended in growth medium and plated on the appropriate tissue culture dish.
After electroporation, cells were either plated directly on multiwell plates for harvest at short time periods (typically 4 days or less) or on tissue culture dishes for trypsinization and seeding onto multiwell plates one or two days before final harvest for longer time periods.
For immunofluorescent detection of dengue-specific proteins, cells growing on chamber slides were rinsed in room-temperature PBS and then fixed in cold acetone for 10 min at -20°C. After being air dried, each chamber was covered with 50 ul of a 1:50 dilution of DEN2-specific hyperimmune mouse ascitic fluid (HMAF, American Type Culture Collection) in PBS plus 2% normal goat serum and incubated at room temperature for 1 h in a humidified atmosphere and then rinsed twice in PBS. After washing, cells were subsequently incubated with a 1:100 dilution of fluorescein isothiocyanate-labeld goat anti-mouse antibodies (Kirkegaard and Perry Laboratory) and rinsed twice in PBS. For detection of HIV-specific proteins, the same protocol was used except that cells were initially incubated with human HIV-1 serum from Waldheim Pharmazeutika Ges.m.b.H. Neufeld-Vienna, Austria and then subsequently incubated with fluorescent-labeled goat anti-human antibody. Cells in some, but not all experiments were counterstained with 0.02% Evans Blue.
For dual labeling, the first antibodies were a 1:50 dilution of dengue type 2 specific hyperimmune mouse ascitic fluid (HMAF, American type culture collection) and a 1:100 dilution of human HIV positive serum in PBS plus 2% normal goat serum. The second antibodies were a 1:100 dilution of FITC-labeled goat anti-human antibodies (Waldeim Pharmazeutika) and a 1:50 dilution of goat anti-mouse IgG-L-Rhodamine (Boehringer Mannheim Biochemicals).
We thank Dr. Owen Wood for help with immunofluorescence photomicrography.
- Nathanson N, Mathieson BJ: Biological Considerations in the Development of a Human Immunodeficiency Virus Vaccine. Journal of Infectious Diseases. 2000, 182: 579-589. 10.1086/315707.View ArticlePubMedGoogle Scholar
- Cho MW: Assessment of HIV Vaccine Development: Past, Present, and Future. Advances in Pharmacology. 2000, 49: 263-314.View ArticlePubMedGoogle Scholar
- Clerici M, Giorgi JV, Chou CC: Cell-mediated immune response to human immunodeficiency virus (HIV) type 1 in seronegative homosexual men with recent sexual exposure to HIV-1. J Infect Dis. 1992, 165: 1012-1019.View ArticlePubMedGoogle Scholar
- Kaul R, Kimani J, Dong T: Late seroconversion in HIV "resistant" Nairobi prostitutes is associated with a preceding decrease in HIV exposure. [abstract 489]. In: Program and abstracts of the 7th Conference on Retroviruses and Opportunistic Infections (San Francisco). San Francisco: Foundation for Retrovirology and Human Health,. 2000, 168-Google Scholar
- Baba TW, Liska V, Khimani AH, Ray NB, Dailey PJ, Penninck D, Bronson R, Greene MF, McClure HM, Martin LN, Ruprecht RM: Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques. Nat. Med. 1999, 5: 194-203. 10.1038/8859.View ArticlePubMedGoogle Scholar
- Baba TW, Jeong YS, Pennick D, Bronson R, Greene MF, Ruprecht RM: Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science. 1995, 267: 1820-1825.View ArticlePubMedGoogle Scholar
- Bogers WM, Niphuis H, ten Haaft P, Laman JD, Koornstra W, Heeney JL: Protection from HIV-1 envelope-bearing chimeric simian immunodeficiency virus (SHIV) in rhesus macaques infected with attenuated SIV: Consequences of challenge. AIDS. 1995, 9: F13-F18.PubMedGoogle Scholar
- Greenough TC, Sullivan JL, Desrosiers RC: Declining CD4 T-cell counts in a person infected with nef-deleted HIV-1. N. Engl. J. Med,. 1999, 340: 236-237. 10.1056/NEJM199901213400314.View ArticleGoogle Scholar
- Berkhout B, Verhoef K, van Wamel JL, Back NK: Genetic instability of live, attenuated human immunodeficiency virus type 1 vaccine strains. J. Virol. 1999, 73: 1138-1145.PubMed CentralPubMedGoogle Scholar
- Rose NF, Marx PA, Luckay A, Nixon DF, Moretto WJ, Donahoe SM, Montefiori D, Roberts A, Buonocore L, Rose JK: An Effective AIDS Vaccine Based on Live Attenuated Vesicular Stomatitis Virus Recombinants. Cell. 2001, 106: 539-549.View ArticlePubMedGoogle Scholar
- Khromykn AA, Westaway ED: Sub-genomic replicons of the flavivirus Kunjin: construction and applications. J. Virol. 1997, 71(2): 1497-1505.Google Scholar
- Pang X, Zhang M, Dayton AI: Development of dengue virus type 2 replicons capable of prolonged expression in host cells. BMC Microbiology. 2001, 1: 18-10.1186/1471-2180-1-18.PubMed CentralView ArticlePubMedGoogle Scholar
- Reitter J, Means R, Desrosiers RC: A role for carbohydrates in immune evasion in AIDS. Nat Med. 1998, 4: 679-684.View ArticlePubMedGoogle Scholar
- Chan DC, Kim PS: HIV entry and its inhibition. Cell. 1998, 93: 681-684.View ArticlePubMedGoogle Scholar
- LaCasse RA, Follis KE, Trahey M: Fusion-competent vaccines: broad neutralization of primary isolates of HIV. Science. 1999, 283: 357-362. 10.1126/science.283.5400.357.View ArticlePubMedGoogle Scholar
- Cooney EL, Collier AC, Greenberg PD, Coombs RW, Zarling J, Arditti DE, Hoffman MC, Hu SL, Corey L: Safety of and immunological response to a recombinant vaccinia virus vaccine expressing HIV envelope glycoprotein. Lancet. 1991, 337(8741): 567-572. 10.1016/0140-6736(91)91636-9.View ArticleGoogle Scholar
- Cooney EL, McElrath MJ, Corey L, Hu SL, Collier AC, Arditti D, Hoffman M, Coombs RW, Smith GE, Greenberg PD: Enhanced immunity to human immunodeficiency virus (HIV) envelope elicited by a combined vaccine regimen consisting of priming with a vaccinia recombinant expressing HIV envelope and boosting with gp160 protein. Proc Natl Acad Sci U S A. 1993, 90(5): 1882-1886.View ArticleGoogle Scholar
- Graham BS, Belshe RB, Clements ML, Dolin R, Corey L, Wright PF, Gorse GJ, Midthun K, Keefer MC, Roberts NJ: Vaccination of vaccinia-naive adults with human immunodeficiency virus type 1 gp160 recombinant vaccinia virus in a blinded, controlled, randomized clinical trial. The AIDS Vaccine Clinical Trials Network. J Infect Dis. 1992, 166(2): 244-52.View ArticleGoogle Scholar
- McElrath MJ, Corey L, Berger D, Hoffman MC, Klucking S, Dragavon J, Peterson E, Greenberg PD: Immune responses elicited by recombinant vaccinia-human immunodeficiency virus (HIV) envelope and HIV envelope protein: analysis of the durability of responses and effect of repeated boosting. J Infect Dis. 1994, 169(1): 41-47.View ArticleGoogle Scholar
- Monath TP: Dengue: the risk to developed and developing countries. Proc Natl Acad Sci USA. 1994, 91: 2395-2400.PubMed CentralView ArticlePubMedGoogle Scholar
- Morens DM: Antibody-dependent enhancement of infection and the pathogenesis of viral disease. Clinical Infectious Diseases. 1994, 19: 500-512.View ArticlePubMedGoogle Scholar
- Halstead SB: Pathogenesis of dengue: Challenges to molecular biology. Science. 1988, 239: 476-481.View ArticlePubMedGoogle Scholar
- Chambers TJ, Chang HS, Galler R, Rice CM: Flavivirus Genome Organization, Expression and Replication. Ann Rev Microbiol. 1990, 44: 649-6888. 10.1146/annurev.micro.44.1.649.View ArticleGoogle Scholar
- Rice CM: Flaviviridae: the virus and their replication. In: Fields Virology 3rd ed. Philadelphia, Pa. Lippincott-Raven Publishers,. 1996, 931-996.Google Scholar
- Thomas CJ, Hahn CS, Galler R, Rice CM: Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990, 44: 649-88. 10.1146/annurev.micro.44.1.649.View ArticleGoogle Scholar
- Heinz FX: Epitope mapping of flavivirus glycoproteins. Adv Virus Res. 1986, 31: 103-168.View ArticlePubMedGoogle Scholar
- Bhamarapravati N, Sutee Y: Live attenuated tetravalent dengue vaccine. Vaccine. 2000, 18 Suppl 2: 44-47. 10.1016/S0264-410X(00)00040-2.View ArticlePubMedGoogle Scholar
- Kanesa-thasan N, Sun W, Kim-Ahn G, Van Albert S, Putnak JR, King A, Raengsakulsrach B, Christ-Schmidt H, Gilson K, Zahradnik JM, Vaughn I, Innis BL, Saluzzo JF, Hoke CH: Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine. 2001, 19(23-24): 3179-3188. 10.1016/S0264-410X(01)00020-2.View ArticleGoogle Scholar
- Schlesinger JJ, Brandriss MW, Walsh EE: Immunization with the dengue 2 virus nonstructural glycoprotein NS1. J. Gen. Virol. 1987, 68: 853-857.View ArticlePubMedGoogle Scholar
- Falgout B, Bray M, Schlesinger JJ, Lai C-J: Immunization of Mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis. J. Virol. 1990, 64(9): 4356-4363.PubMed CentralGoogle Scholar
- Henchal EA, Henchal LS, Schlesinger JJ: Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challenge with dengue 2 virus. J Gen Virol. 1988, 69 (Pt 8): 2101-2107.View ArticleGoogle Scholar
- Brinton MA, Kurane I, Mathew A, Zeng L, Shi PY, Rothman A, Ennis FA: Immune mediated and inherited defenses against flaviviruses. Clin Diagn Virol. 1998, 10(2-3): 129-39. 10.1016/S0928-0197(98)00039-7.View ArticleGoogle Scholar
- Dittmer U, Brooks DM, Hasenkrug KJ: Protection against establishment of retro viral persistence by vaccination with a live attenuated virus. J Virol. 1999, 73: 3753-3757.PubMed CentralPubMedGoogle Scholar
- Heeney J, Akerblom L, Barnett S: HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates. Immunol Lett. 1999, 66: 189-95. 10.1016/S0165-2478(98)00157-6.View ArticlePubMedGoogle Scholar
- Hirsch V, Fuerst T, Sutter G: Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara. J Virol. 1996, 70: 3741-3752.PubMed CentralPubMedGoogle Scholar
- Quesada-Rolander M, Makitalo B, Thorstensson R: Protection against mucosal SIVsm challenge in macaques infected with a chimeric SIV that expresses HIV type 1 envelope. AIDS Res Hum Retroviruses. 1996, 12: 993-999.View ArticlePubMedGoogle Scholar
- Letvin NL, Montefiori D, Yasutami Y: Potent, protective anti-HIV immune response generated by bimodal HIV envelope DNA plus protein vaccination. Proc Natl Acad Sci USA. 1997, 94: 9378-383. 10.1073/pnas.94.17.9378.PubMed CentralView ArticlePubMedGoogle Scholar
- Miller CJ, McChesney MB, Lu X: Rhesus macaques previously infected with simian/human immunodeficiency virus are protected from vaginal challenge with pathogenic SIVmac239. J Virol. 1997, 71: 1911-1921.PubMed CentralPubMedGoogle Scholar
- Shibata R, Siemon C, Czajak SC: Live, attenuated simian immunodeficiency virus vaccines elicit potent resistance against a challenge with a human immunodeficiency virus type 1 chimeric virus. J Virol. 1997, 71: 8141-8148.PubMed CentralPubMedGoogle Scholar
- Gundlach B, Reiprich S, Sopper S: Env-independent protection induced by live, attenuated simian immunodeficiency virus vaccines. J Virol. 1998, 72: 7846-7251.PubMed CentralPubMedGoogle Scholar
- Girard M, Habel A, Chanel C: New prospects for the development of a vaccine against human immunodeficiency virus type 1. An overview. C R Acad Sci III. 1999, 322(11): 959-966.View ArticleGoogle Scholar
- Barnett SW, Lu S, Srivastava I, Cherpelis S, Gettie A, Blanchard J, Wang S, Mboudjeka I, Leung L, Lian Y, Fong A, Buckner C, Ly A, Hilt S, Ulmer J, Wild CT, Mascola JR, Stamatatos L: The ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region. J Virol. 2001, 75(12): 5526-5540. 10.1128/JVI.75.12.5526-5540.2001.PubMed CentralView ArticleGoogle Scholar
- Polo S, Ketner G, Levis R, Falgout B: Infectious RNA Transcripts from full-length dengue virus type 2 cDNA clones made in Yeast. J Virol. 1997, 71(7): 5366-5374.PubMed CentralGoogle Scholar
- Pur B, Polo S, Hayes CG, Falgout B: Construction of a full length infection clone for dengue-1 virus western pacific 74 strain. Virus Genes. 2000, 20(1): 57-63. 10.1023/A:1008160123754.View ArticleGoogle Scholar
- Spencer F, Ketner G, Connelly C, Hieter P: Targeted recombination-based cloning and manipulation of large DNA segments in yeast. Methods Companion Methods Enzymol. 1993, 5: 161-175. 10.1006/meth.1993.1021.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.