| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | September 24, 2002 |
| PATENT TITLE |
Dengue nucleic acid vaccines that induce neutralizing antibodies |
| PATENT ABSTRACT | A vaccine for promoting an immune response in a mammalian subject includes a eucaryotic plasmid expression vector which include at least part of the envelope gene and optionally, the PreM gene of dengue virus. In order to minimize immune enhancement, vaccines of up to the four serotypes of dengue are combined in a single vaccine. The vaccine in a suitable pharmaceutical carrier constitutes a pharmaceutical composition which is injected into the subject |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 4, 1997 |
| PATENT REFERENCES CITED |
Phillpotts, R.J. et al. Arch. Virol. 141: 743-749, 1996.* Bray, M. et al. Virology. 185 (1): 505-508, 1991.* Kochel; Inoculation of Plasmids Expressing The Dengue-2 Envelope Gene Elicit Neutralizing Antibodies in Mice; Vaccine vol. 5. pp 547-552 1997. Cox; Bovine Herpesvirus 1: Immune Responses in Mice And Cattle Injected with Plasmid DNA; Journal of Virology Sep. 1993; vol. 97, pp. 5664-5667. Sedegah; Protection Against Malaria by Immunization with Plasmid DNA Encoding Circumsporozoite Protein; Proc. Natl, Acad. Sci,.; Oct. 1994; vol 91, pp. 9866-9870. Major; DNA Based Immunization with Chimeric Vectors for the Induction of Immune Responses Against the Hepatitis C Virus Mucleocapsid; Journal of Virology; Sep. 1995; pp. 5798-5805. Wang; Gene Inoculation Generates Immune Responses Against Human Immunodeficiency Virus Type 1; Proc. Natl. Acad. Sci.; May 1993; vol. 90, pp. 4156-4160. Wolff; Direct Gene Transfer into Mouse Muscle in Vivo; Science vol.; Mar. 23, 1990; 1465-1468. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A pharmaceutical composition capable of inducing an immune response in a mammalian subject, comprising an immunogenic amount of a eukaryotic plasmid expression vector in pharmaceutically acceptable form, wherein said plasmid expression vector is functional in mammalian subjects and includes preM and at least 92% of the envelope gene of a dengue W virus, where W is a number selected from the group consisting of 1, 2, 3 and 4. 2. The pharmaceutical composition of claim 1 further comprising a second plasmid including the PreM and at least 92% of the envelope gene of dengue X virus, where X is a number different from W and is selected from a the group consisting of 1, 2, 3 and 4. 3. The pharmaceutical composition of claim 2 further comprising a third plasmid including the PreM and at least 92% of the envelope gene of dengue Y virus, where Y is a number different from W and from X and is selected from the group consisting of 1, 2, 3 and 4. 4. The pharmaceutical composition of claim 3 further comprising a fourth plasmid including the PreM and at least 92% of the envelope gene of dengue Z virus, where Z is a number different from W, from X, and from Y, and is selected from the group consisting of 1, 2, 3 and 4. 5. The pharmaceutical composition of claim 1, further comprising a suitable pharmaceutical carrier. 6. The pharmaceutical composition of claim 5, which is in injectable form. 7. The pharmaceutical composition of claim 1, which is a vaccine capable of inducing a protective immune response in said mammalian subject, comprising an immunoprotective amount of said plasmid expression vector. 8. The vaccine of claim 7, which comprises a suitable pharmaceutical carrier, and is in injectable form. 9. A method of inducing an immune response in a mammalian subject, comprising the step of injecting the composition of claim 6. 10. A method of inducing a protective immune response in a mammalian subjects comprising the step of injecting the vaccine of claim 8. 11. The pharmaceutical composition of claim 2, further comprising a suitable pharmaceutical carrier. 12. The pharmaceutical composition of claim 11, which is in injectable form. 13. The pharmaceutical composition of claim 2, which is a vaccine capable of inducing a protective immune response in said mammalian subject, comprising an immunoprotective amount of said plasmid expression vector. 14. The vaccine of claim 13, which comprises a suitable pharmaceutical carrier, and is in injectable form. 15. A method of inducing an immune response in a mammalian subject, comprising the step of injecting the composition of claim 12. 16. A method of inducing a protective immune response in a mammalian subject comprising the step of injecting the vaccine of claim 14. 17. The pharmaceutical composition of claim 3, further comprising a suitable pharmaceutical carrier. 18. The pharmaceutical composition of claim 17, which is in injectable form. 19. The pharmaceutical composition of claim 3, which is a vaccine capable of inducing a protective immune response in said mammalian subject, comprising an immunoprotective amount of said plasmid expression vector. 20. The vaccine of claim 19, which comprises a suitable pharmaceutical carrier, and is in injectable form. 21. A method of inducing an immune response in a mammalian subject, comprising the step of injecting the composition of claim 18. 22. A method of inducing a protective immune response in a mammalian subject, comprising the step of injecting the vaccine of claim 20. 23. The pharmaceutical composition of claim 4, further comprising a suitable pharmaceutical carrier. 24. The pharmaceutical composition of claim 23, which is in injectable form. 25. The pharmaceutical composition of claim 4, which is a vaccine capable of inducing a protective immune response in said mammalian subject, comprising an immunoprotective amount of said plasmid expression vector. 26. The vaccine of claim 25, which comprises a suitable pharmaceutical carrier, and is in injectable form. 27. A method of inducing an immune response in a mammalian subject, comprising the step of injecting the composition of claim 24. 28. A method of inducing a protective immune response in a mammalian subject, comprising the step of injecting the vaccine of claim 26. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to nucleic acid vaccines and more specifically to Dengue nucleic acid vaccines. BACKGROUND OF THE INVENTION Dengue (Den) viruses belong to the flavivirus genus of the family Flaviviridae and are of four serotypes, Den 1-4. Dengue viruses are positive strand RNA viruses which code for ten genes. The genes are translated as a polyprotein which is cleaved by host and viral proteinases. The virus envelope (E) protein is the major antigen against which neutralizing antibodies are directed. These antibodies have been shown to be capable of protecting against dengue virus infection.sup.1. The membrane protein also appears on the virion surface and is required for the proper processing of E. Dengue viruses are transmitted primarily by the mosquito, Aedes aegypti, and are a major cause of morbidity and mortality throughout tropical and subtropical regions worldwide.sup.2. It is estimated that there are over 100 million cases, annually, of dengue fever.sup.3. Human dengue illnesses range from an acute undifferentiated fever to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). A primary infection usually causes dengue fever. The illness is generally mild and the person apparently acquires a life long immunity against the serotype of dengue virus causing the infection. However, if a person acquires a second dengue infection with a different serotype, the illness may be more severe and lead to hemorrhagic fever or shock syndrome, with a mortality rate between one and five percent. The increased severity of the secondary infection is caused by an immune enhancement phenomenon.sup.4. Immune enhancement begins when non-neutralizing antibodies, generated from the earlier infection with one dengue serotype, bind to but do not neutralize the virus causing the secondary infection. The Fc portion of the antibody in the virus-antibody complexes then binds to the Fc receptors present on mononuclear phagocytes of the immune system, resulting in enhanced infection of these cells. The enhanced infection leads to the release of cytokines that ultimately cause capillary leak and coagulopathy, the principal pathogenic mechanisms in DHF/DSS. At present there is no approved vaccine for dengue viruses. The most effective dengue vaccine would elicit sustained protective levels of neutralizing antibodies against all four serotypes so as to avoid the possibility of immune enhancement in a vaccinated individual who might become secondarily infected with a different epidemic or endemic dengue of a different serotype. Work in mice and primates with inactivated whole virus and at recombinant protein dengue vaccines has generally been disappointing because of the lack of sustained protective neutralizing antibodies induced. Vaccination with live attenuated dengue virus vaccines is another promising approach, but difficulties still remain in developing a product that is immunogenic and does not cause dengue fever-like side effects. DNA vaccines for dengue will offer substantial advantages over these other approaches in that sustained immunity can be achieved without the risk of dengue fever-like side effects or immune-enhancement. A description of DNA inoculation was presented by Vical, Inc. (San Diego, Calif.).sup.5. Vical demonstrated that one could inject a gene, (in the proper context of an eukaryotic expression vector), into the muscle of an animal and that the injected gene would be expressed. The expression vector must contain the proper eukaryotic transcriptional regulatory elements (promoter/enhancer and polyadenylation site) and a multiple cloning site so that once inside the cell of the subject the gene will be expressed. Once the gene is transcribed it is translated and processed to its mature protein product. The basic DNA injection system offers great potential for vaccine development. One clones the genes which contain the desired antigenic determinates into an expression construct and injects the DNA into an animal. Since the proteins are translated and processed within the host cells, proper conformation of B cell epitopes on secreted proteins and induction of class 1 MHC-dependent immune responses should occur appropriately. The technique of DNA injection is being used to develop vaccines against many pathogens including: Influenza.sup.6, HIV.sup.7, Hepatitis C.sup.8, Malaria.sup.9 and Herpes.sup.10. SUMMARY OF THE INVENTION It is an object of this invention to protect a subject or community against infection by dengue virus. It is another object of this invention to provide protection against infection by dengue virus by using a nucleic acid vaccine. It is an object of this invention to provide protection against more than one dengue virus serotype. These and additional objects of the invention are accomplished by taking the envelope (E) and optionally, the membrane (PreM) genes of dengue virus, serotypes 1, 2, 3, and 4 and cloning them into eukaryotic plasmid expression vectors. The resultant plasmid DNAs (dengue DNA vaccines) are injected into a mammalian subject where in vivo the E proteins are expressed and subsequently recognized and processed by immune cells. This results in the generation of humoral and cellular immunity that is protective against dengue virus infections of each of the four serotypes. We have used the DNA injection technology to develop DNA vaccines against the Den viruses. We have cloned genes from each of the four Den serotype viruses into expression vectors and have assessed their potential as Den virus vaccines. The Den genes which we have incorporated into our DNA vaccines include: 80-100% of the E gene; either alone or expressed with the PreM gene. The choice of these genes systems from publications which demonstrate that: E contains the virus' major antigenic determinants.sup.11 ; not all of E is required to obtain a protective immune response.sup.12 ; and PreM affects the conformation of the produced E protein.sup.13. As Den virus DNA vaccines, some combinations of the above-listed genes yield better immune responses than others. These and other objects, features and advantages of the present invention are described in or are apparent from the following detailed description of preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS The invention will be described with reference to the drawings, in which like elements have been denoted throughout by like reference numerals. The representations in each of the figures is diagrammatic and no attempt is made to indicate actual scales or precise ratios. Proportional relationships are shown as approximations. FIG. 1 is a graph showing time response to inoculation with two different Den type 2 DNA vaccines. FIG. 2 is a graph showing the time course of plaque reduction neutralization test titers from sera of mice inoculated with p1012D2ME against Den type 2. FIG. 3 is a bar graph showing the response to inoculation with various dosages of Den type 2 DNA vaccine +/- Immuno-stimulatory DNA sequences. FIG. 4 is a bar graph showing the response to inoculation with suboptimal dose of p1012D2ME and various amounts of Immuno-stimulatory DNA sequences (puC). FIG. 5 is a Kaplan Meier Survival plot summarizing the results of a mouse challenge experiment using the dengue type 2 DNA vaccine. FIG. 6 is a graph showing the immunogenicity of a Den 1 DNA vaccine containing 80% E. FIG. 7 is a graph illustrating the immunogenicity of DNA vaccines constructed against dengue serotype 1 and containing the PreM gene and different amounts of the E gene. FIG. 8 is a graph illustrating the immunogenicity of DNA vaccines constructed against dengue serotype 2 and against dengue serotype 3. DETAILED DESCRIPTION OF THE INVENTION By this invention the newly developed nucleic acid vaccination approach is adapted to provide a dengue virus vaccine, also called a naked DNA dengue vaccine. Eukaryotic plasmid expression vectors containing the PreM and at least part of the E gene of a virus selected from the group consisting of dengue-1 virus, dengue-2 virus, dengue-3 virus and dengue-4 virus are formed. The constructs are then injected into a mammalian subject; either individually or in combinations including up to all four serotypes. Nucleic acid vaccines possess many of the advantages of live-attenuated vaccines without the concern of inducing clinical illness by the vaccine itself. Since the viral proteins are translated and processed within the host cells, proper conformation of epitopes on secreted proteins and induction of class I MHC-dependent immune responses occur appropriately, and induction of both humoral and cellular immunity are long lasting. The invention applies nucleic acid vaccine technology to develop a DNA plasmid construct that elicits anti-dengue neutralizing antibodies in a mouse model which leads to and is adaptable to later application to other mammals, including non-human and human primates. In general, the naked DNA dengue vaccine of the present invention includes the dengue preM gene, and at least part of the dengue E gene, for dengue type 1, 2, 3 or 4, or some combination of types 1, 2, 3 and 4. The published sequences for the preM and E genes of dengue serotype 1, West Pacific strain, serotype 2, New Guinea C strain, serotype 3, H87 strain, and serotype 4, H81 strain, are referred to herein as sequence id. nos. 4, 1, 5 and 6, respectively. The vaccine preferably includes at least about 80% and most preferably, about 100%, about 92% or about 80% of the dengue E gene. Alternatively, the vaccine of the present invention preferably includes about 100%, about 92% or about 80% of the dengue E gene and need not include any of the preM gene. Silent base substitutions may be made to the above-described DNA without altering the effectiveness or scope of the present invention. The DNA dengue vaccine of the present invention does not include any nucleotides or sequences susceptible to expression in a mammalian cell other than as described above. The expression vector used to produce the above vaccine is a eukaryotic expression vector including a promotor/enhancer, cloning site and termination sequence. The promoter/enhancer works with RNA polymerase to initiate transcription of mRNA and the cloning site is for insertion of the dengue genes of interest. The expression vector preferably also includes an intron, which is not expressed in the protein produced by the mammalian cell but speeds up expression of the genes of interest. The vaccine of the present invention is preferably administered by intradermal or intramuscular injection but any other means of administration known to persons of ordinary skill in the art may also be used. The vaccine is a pharmaceutical composition suitable for injection into a subject. It includes the above-described plasmid in a suitable injectable pharmaceutical carrier that does not interact with the plasmid. Examples of suitable carriers for the vaccine of the present invention are phosphate buffered saline (PBS) and normal saline (150 mM sodium chloride). Experimental Results This invention shows that the nucleic acid vaccination technique can be used to raise a protective anti-dengue virus immune response in mice. In the first series of experiments, two different plasmid expression vectors, pkCMVintPolyli and pVR1012 were used. A detailed description of pkCMVintPolyli and pVR1012 is given below under "Examples". Briefly, pkCMVintPolyli (developed by Vical, Inc.) is an expression vector which contains the CMV enhancer and CMV intron A, a multiple cloning site, the SV40 poly A site (termination sequence) and the SV40 origin of replication. The vector pVR1012, developed by Vical, Inc. and referred to herein as p1012, is an expression vector which contains the CMV enhancer, the CMV intron A sequence, a multiple cloning site, and the Bovine Growth Hormone termination sequence. p1012 does not include the SV40 origin of replication. The Den-2 genes PreM and 92% E were cloned into pkCMVintPolyli and p1012 (see "Examples" below for cloning details); the resultant constructs are pD2ME and p1012D2ME, respectively. The latter construct is also referred to herein as p1012D2ME92, the sequence of which is listed as sequence id. no. 2. To determine if pD2ME and p1012D2ME properly express the E gene in vitro, Immune Fluorescence Assays (IFA) and Radio-labeled Immune Precipitation Analysis (RIPA) were performed on transiently transfected cells. Details of IFA, RIPA and transfections are provided below under "Examples" and the results of these measurements are shown in Table I. TABLE I Immune Fluorescence Assay of Construct Transfected Cells Antisera Construct Den-2 HIAF 4G2 3H5 pkCMVintPolyli - - - pVR1012 - - - PD2ME + + + + + + p1012D2ME + + + + + + + + + Adenovirus transformed human embryonic kidney cells (293 cells) were transfected with vector only (pkCMVintPolyli or pVR1012) or Den type-2 DNA vaccine (pD2ME or p1012D2ME). Forty eight hours post transfection the cells were scraped off their plates and spotted onto slides. The slides were then reacted with either Den-2 hyperimmune ascitic fluid (HIAF) or conformation specific anti-Den envelope antibodies 4G2 and 3H5. Fluorescein conjugated # secondary antibody was used to visualize the primary antibody-antigen interaction by fluorescence. The slides were rated on a fluorescence scale of - (negative) to 4+ (four positive). Table I shows that cells transfected with either pD2ME or p1012D2ME express the truncated E protein of Den-2. The ability of Den-2 Hyper-Immune Ascitic Fluid (HIAF) to recognize the truncated E protein in the IFA demonstrates the expression of the truncated E protein. No truncated E protein is expressed in cells transfected with vector only (i.e. pkCMVintPolyli or p1012). The proper expression of the truncated E gene, and retention of conformational epitopes, in vitro, is demonstrated by the ability of two, Den-2, conformation-dependent monoclonal antibodies, 4G2 and 3H5, to specifically recognize the protein. Table I also shows that genes cloned into pVR1012 yield greater amounts of fluorescence than the same genes cloned into pkCMVintPolyli, thus suggesting that genes cloned into p1012 are expressed at higher levels than the same genes cloned into pkCMVintPolyli, in vitro. This observation was also seen in RIPA of identically transfected cells. Den-2 HIAF, 3H5 and 4G2 each immune precipitated greater amounts of the truncated E protein from cell lysates of p1012D2ME transfected cells than from pD2ME-transfected cells. RIPA also detected truncated E protein in the media of transfected cells demonstrating that the truncated E protein is secreted (data not shown). To determine if pD2ME or p1012D2ME could induce the production of anti-Den antibodies in an in vivo system, mice were inoculated with the DNA constructs (detailed description of DNA inoculation and in vitro analysis of immune responses is presented below under "Examples"). Groups of ten three-week old mice were intradermally inoculated with either pVR1012, pkCMVintPolyli, pD2ME or p1012D2ME (on day 0) and boosted on days 9, 22 and 57 post priming. Sera were collected from the mice on days 22, 57 and 92 post priming (and day 154 for p1012D2ME inoculated mice) and assayed for the presence anti-Den antibodies by enzyme-linked immunosorbent assay (ELISA). Referring now to FIG. 1 the time course of antibody production is shown. The three week old mice that were intradermally inoculated with pD2ME or p1012D2ME produced dengue antibodies, as manifested by ELISA results. Mice inoculated with pkCMVintPolyli or pVR10122 did not produce any Den antibodies. The mean ELISA optical density (OD) obtained from sera of mice inoculated with p1012D2ME was two-fold higher, after the third DNA boost, than that obtained from mice inoculated with pD2ME. It is believed that this phenomenon is due to the genes cloned into pVR1012 which shows a higher level of expression as compared with genes cloned into pkCMVintPolyli. To determine if the Den-2 antibodies produced by pD2ME and p1012D2ME inoculated mice contain virus neutralizing activity, Plaque Reduction Neutralization Tests (PRNT) were performed, on day 92 (post priming) sera. The PRNT.sub.50 for the sera from pD2ME and p1012D2ME inoculated mice are shown in Table II. Day 92 sera from mice inoculated with pkCMVintPolyli or p1012 did not produce Den-2 antibodies (See FIG. 1), nor did day 92 sera from those mice contain any Den-2 virus neutralizing activity (data not shown). All sera from mice inoculated with either pD2ME or p1012D2ME contain virus neutralizing activity. The mean reciprocal dilution titer for sera from mice immunized with pD2ME is 48. The mean reciprocal dilution titer for sera from p1012D2ME immunized mice is 195. The PRNT.sub.50 titers for sera from p1012D2ME vaccinated mice are four times higher than those obtained from sera of pD2ME vaccinated mice. This result is not unexpected since the p1012D2ME vaccinated mice produced higher levels of Den-2 antibodies (as manifested by ELISA, See FIG. 1). The neutralizing titers obtained from both sets of mice are comparable to those titers previously shown in the literature to be sufficient to protect a mouse from a lethal challenge of Den virus. Mice immunized with Den virus produce PRNT.sub.50 reciprocal titers as high as 360.sup.14 and other Den virus vaccines have been shown to be protective against lethal challenge with titers as low as ten.sup.15. Thus, sera from the p1012D2ME vaccinated mice are producing PRNT.sub.50 titers approaching that of Den virus. TABLE II Neutralizing antibody titers in mice immunized with p1012D2ME or pD2ME. p1012D2ME Sera pD2ME Sera Animal # PRNT.sub.50 Animal # PRNT.sub.50 1 1:320 1 1:40 2 1:80 2 1:40 3 1:160 3 1:20 4 1:80 4 1:80 5 1:320 5 1:40 6 1:320 6 1:80 7 1:10 7 1:80 8 1:20 8 1:40 9 1:320 9 1:20 10 1:320 10 1:40 A desirable feature of a Den virus vaccine is that it be capable of affording sustained protection. The indicator of protection that we are using is PRNT.sub.50 titers. The day 92 sera from p1012D2ME vaccinated mice contained mean reciprocal PRNT.sub.50 titers of 195. Sera were collected from those same mice on day 154 (post priming) and assayed by ELISA for the presence of Den-2 antibodies and for neutralizing titers by PRNT.sub.50. FIG. 2 shows that the mice were still producing high levels of Den-2 antibodies. The mean reciprocal PRNT.sub.50 titers were still 190, demonstrating the sustained protective efficacy of our Den-2 DNA vaccine. Neutralizing antibodies are an indicator of the protective efficacy of a vaccine, but direct protection from a lethal challenge of virus unequivocally demonstrates the efficacy of the vaccine. In the murine system, the standard dengue virus challenge is conducted with six week old mice since older mice are less susceptible to the virus.sup.16. Three week old mice are immunized and challenged three weeks later. Ten mice were inoculated with p1012D2ME and challenged at six weeks of age (day 21 post priming), but protection was not observed. That result was not completely unexpected since in our system the mice did not produce significant amounts of Den antibodies at the time of challenge but did produce significant amounts of Den antibodies later (See, FIG. 1). In light of the above results, an earlier antibody response would be desirable. Therefore, the use of immuno-stimulatory DNA sequences (ISS) and the effects of lower dosages when using ISS were explored. Groups of 5 mice were immunized ID with either 200 .mu.g, 50 .mu.g, 12.5 .mu.g or 3.1 .mu.g of the dengue 2 DNA vaccine p1012D2ME92. Additional groups of mice were co-immunized with the same amounts of vaccine together with 100 .mu.g of the commercially available pUC 19 plasmid (Life Technologies/Gibco). The rationale for using pUC 19 is that in studies where plasmids coding for the protein beta galactosidase were co-injected with pUC 19, both cellular and humoral immunity to this protein in mice were enhanced.sup.17. The plasmid pUC 19 was found to contain two copies of the ISS (--AACGTT--) located in the ampicillin resistance gene. For these dosing studies, mice were primed on day 0 and boosted on days 10 and 25 and serum samples obtained on days 25 and 38. At day 25 (after a single boost) the dengue antibody response, as measured by mean OD value, in mice that received 3.1 .mu.g and 12.5 .mu.g of p1012D2ME was greater in the pUC group but the differences were not statistically significant. Referring now to FIG. 3, mice immunized with 50 .mu.g and 200 .mu.g of DNA vaccine without pUC had a significantly greater mean OD value compared to the mice that received the same dose along with pUC (FIG. 3). The detection of dengue antibody at day 25 in the 200 .mu.g group contrasts earlier findings where dengue antibodies were not seen until approximately two months post-priming. The reason for this difference is not clear, but may be the result of improved inoculation techniques. By day 38, the mean OD values for the pUC group were higher throughout the range of doses compared to OD values for the group that received vaccine alone (FIG. 3). However the only statistically significant difference was seen between the groups that were immunized with 3.1 .mu.g (p<0.05), indicating that pUC significantly enhanced the immunogenicity of the dengue DNA vaccine at the lowest dosage. To further evaluate the immuno-stimulatory effects of pUC on the immune response to 3 .mu.g of DNA vaccine, four groups of five mice each were co-immunized with 3 .mu.g of p1012D2ME92 together with decreasing amounts of pUC (100 .mu.g, 20 .mu.g, 4 .mu.g, and 0 .mu.g). The mice were primed and then boosted on days 10 and 21. As shown in FIG. 4, serum samples obtained on day 21 (after the first boost) and on day 50 (after the second boost) showed a significantly greater antibody response, as measured by the mean ELISA OD, in the group of animals co-immunized with 100 .mu.g of pUC. A dose-related decrease in mean OD was observed with a minimal antibody response occurring in the group of animals immunized with 3 .mu.g of vaccine alone. Further analysis of the day 57 serum samples using the plaque reduction neutralization test showed that the mean neutralizing antibody titer in the group co-immunized with 100 .mu.g pUC (1:48) was significantly greater (p<0.008) than the mean titer of the 4 .mu.g (1:24) and 20 .mu.g (1:14) groups. No neutralizing antibodies were detected inthe group immunized with vaccine alone. These results indicate that ISS contained in pUC significantly enhances the neutralizing antibody response of the dengue DNA vaccine and that 100 .mu.g of pUC 19 produced the greatest immunostimulatory effect. The protective efficacy in a mouse intracerebral challenge model using p1012D2ME92 with the co-inoculation of pUC 19 was evaluated. A more detailed description of the challenge can be found below under "Examples". Four groups of ten three-week old mice each were immunized with either 12.5 .mu.g pVRO112 alone, 12.5 .mu.g pVR1012 plus 100 .mu.g pUC 19, 12.5 .mu.g p1012D2ME92 plus 100 .mu.g pUC 19 or 50 .mu.g p1012D2ME92 alone. Mice were primed on day 0, boosted on day 10 post-priming and challenged on day 21 (when mice reached the appropriate challenge age of six weeks). Survival in the groups that received vaccine with or without pUC 19 was 60% compared to 20% and 10% in the pVR1012 only and pVR1012 plus pUC groups, respectively. Because of the small numbers in each group, only the comparison between the vaccine plus pUC 19 and the pVR1012 plus pUC groups showed a statistically significant difference in survival (Kaplan Meier analysis, p<0.05). However, as shown in FIG. 5, if the two vaccine groups and two control groups were combined, the difference in survival was more significant (p=0.005). The above discussion describes Den 2 DNA vaccines. To demonstrate the feasibility of constructing a DNA vaccine against another serotype, 80% of the E gene of Den type 1 virus was cloned into the plasmid vector pkCMVintPolyli; the resultant plasmid is pD1E80 (details of cloning, in vitro, and in vivo experiments are described further below under "Examples"). Proper expression of 80% E off pD1E80, in vitro, was confirmed by IFA and RIPA. In vitro, 80% E was made in cells and secreted from the cells into the cell culture media (data not shown). To determine if pD1E80 could induce an immune response in vivo, three week old mice were inoculated (on day 0) with either pD1E80 or pkCMVintPolyli and boosted on days 9, 21 and 47. Sera were collected from the mice on days 0, 22, 47 and 67 post priming and assayed for the presence of Den-1 antibodies by ELISA. The results are shown in FIG. 6. Mice injected with pkCMVintPolyli did not produce any Den-1 antibodies. Mice that were injected with pD1E80 produced Den-1 antibodies. The time course of antibody production was similar to that seen with the Den-2 DNA vaccines (See, FIG. 1). Day 67 sera were assayed for the presence of neutralizing activity by PRNT. The reciprocal PRNT.sub.50 titers are shown in Table III. Sera from mice injected with pkCMVintPolyli contained no virus neutralizing activity. Sera from mice immunized with pD1E80 contained virus neutralizing activity. The mean reciprocal PRNT.sub.50 titer is 80. Titers at this level should provide significant protection against lethal challenge. TABLE III Neutralizing Antibody Titers in Mice Immunized With pD1E80. Animal # PRNT.sub.50 1 1:40-1:80 2 1:40-1:80 3 1:40-1:80 4 1:80 5 1:40-1:80 6 1:40-1:80 7 1:160 8 1:40-1:80 9 1:40-1:80 10 1:80 The Den-1 80% E gene expressed from pkCMVintPolyli yielded a significant immune response in mice. Since in the dengue 2 experiments genes cloned into the other vector p1012 yielded greater antibody responses than the same genes cloned into pkMVintPolyli, Den 1 genes were cloned into p1012. To evaluate how much of the E gene to include to produce the optimum antibody response, DNA vaccine candidates p1012D1E80, p1012D1ME92, p1012D1ME80 and p1012D1ME100 were prepared. p1012D1E80 has 80% of Den 1 E cloned into p1012; p1012D1ME92 has the PreM and 92% of Den 1 E cloned into p1012; p1012D1ME80 has the PreM and 80% of Den 1 E cloned into p1012 and p1012D1ME100 has the PreM and 100% of Den 1 E cloned into p1012. All constructs were confirmed to express their E gene in vitro by IFA and RIPA (data not shown). To determine if the constructs could induce an immune response in vivo, three week old mice were inoculated (on day 0) with either p1012 or one of the DNA vaccine candidates and boosted on days 10 and 21. Sera were collected from the mice on days 0, 30 and 50 and assayed for the presence of Den-1 antibodies by ELISA. The results are shown in FIG. 7. Mice injected with p1012 did not produce Den-1 antibodies. Mice that were injected with p1012D1ME92 or p1012D1ME80 also did not produce any Den-I antibodies. Mice immunized with p1012D180 or p1012D1ME100 did produce Den 1 antibodies. The DNA vaccine candidate containing the PreM and 100% E elicited the best immune response. The results presented in this application demonstrate the ability of dengue DNA vaccines for dengue types 1 and 2 to elicit dengue antibody responses in mice. The dengue type 2 DNA vaccine was shown to provide significant protection against lethal dengue virus challenge. The neutralizing antibodies produced by the dengue DNA vaccine were shown to be long-lasting. Referring now to FIG. 8, similar DNA vaccine candidates that express the PreM and 100%E genes from Den virus serotype 2 and 3 produce dengue antibodies in mice as measured by ELISA. Mice have been inoculated with similar constructs for dengue 4 and it is expected that these constructs will have similar effects in producing dengue antibodies. A tetravalent dengue DNA vaccine that provides protection against all four serotypes can be prepared by combining the four different DNA vaccines to form a tetravalent mixture or, alternatively, by cloning various combinations of the genes into one or more plasmid vectors. Similarly, the combined vaccine can include 1, 2, 3 or 4 of the individual vaccines or the individual genes. It is expected that such a combined DNA vaccine would provide life-long protection against dengue virus infection without the risk of vaccine induced dengue fever-like side effects or the risk of making vaccinated persons vulnerable to the most severe clinical forms of the disease, DHF/DSS |
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