- Email: [email protected]
DNA vaccination with HIV-1 expressing constructs elicits immune responses in humans
PII: SO264-410X(98)00180-7
Vaccine, Vol. 16, No. 19, pp. 1818-1821, 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410)(/98 $19+0.00
ELSEVIER
DNA vaccination with HIV-l expressing constructs elicits immune responses in humans Kenneth E. Ugen”7, Susan B. Nyland*, Jean D. Bayer?, Cristina Vidal*, Liana Lera”, Sowsan Rasheid”, Michael Chattergoon?, Mark L. Bagarazzil, Richard Ciccarelli§, Terry Higginss, Yaila Baines, Richard Ginsbergs, Rob Roy Macgregor? and David B. Weiner? Humoral and cellular immune responses have been produced by intramuscular vaccination with DNA plasmids expressing HIV1 genes, suggesting possible immunotherapeutic and prophylactic value for these constructs. Vaccination with these constructs has decreased HIV-1 viral load in HIV-l-infected chimpanzees. In addition, naive (i.e. non-HIVl-infected) chimpanzees were protected against a heterologous challenge with HIV-l. Ongoing phase I clinical trials show that therapeutic vaccinations indeed boost anti-HIV-I immune responses in humans. A therapeutic phase I trial on humans with these constructs induced a good safety profile and also demonstrated an immunological potentiation. These findings indicate that further studies with these constructs in humans are warranted. 0 1998 Elsevier Science Ltd. All rights reserved Keywords: Antibody; ELISA; DNA plasmid vaccination
The human immunodeficiency virus type I, HIV-l, continues to challenge researchers due to its ability to escape immune attack and to mutate into multidrug resistant strains. Therefore, the development of effective prophylactic and therapeutic strategies which emphasize enhancement of immunity without increasing the selection pressure for drug resistant mutants is sorely needed. The use of multidrug ‘cocktail’ therapies which include the newer protease inhibitors has reduced the viral load in many patients at the present time. The benefits of an immunoenhancing, non-mutagenic therapeutic system for these individuals might be best realized during the period when viral load is reduced and before multidrug resistant mutants become established. The prevention of future HIV-l infections in high risk populations would require vaccines capable of eliciting immune responses (most likely humoral and cell-mediated). The utility of live attenuated viral preparations against *University of South Florida College of Medicine, Department of Medical Microbiology and Immunology, 12901 Bruce 6. Downs Blvd, Tampa, FL 33612, USA. funiversity of Pennsylvania School of Medicine, Department of Pathology and Laboratory Medicine, 422 Curie Blvd, Room 505, Philadelphia, PA 19104, USA. *Allegheny University of the Health Sciences, Department of Pediatrics, St. Christopher’s Hospital for Children, Front Street and Erie Avenue, Philadelphia, PA 19134, USA. QApollon Inc., Malvern, PA 19355, USA. lITo whom correspondence should be addressed. Tel.: (813) 9741917; Fax: (813) 9744151; E-mail: kugen@coml .med.usf.edu
1818
Vaccine
1998 Volume
16 Number
19
HIV-l has been suggested’. Using this approach for the prevention of HIV-l infection has the obvious advantage of preparing both arms of the immune response for future viral challenge. However, the use of an attenuated HIV-l on high risk individuals who may or may not already be harbouring an undetectable level of virus leads to questions concerning recombination, reversion, and pathogenic expression of new recombinant?. Therefore, vaccine approaches which ‘mimic’ live attenuated preparations without the associated safety concerns are needed. We previously reported on the use of direct nucleic acid inoculation as a novel alternative to live attenuated virus3-‘. This system appears to mimic natural infection in that it may enable potential antigen presenting cells (APC) to process antigenic peptides through MHC I and MHC II pathways, yet is non-infectious. Chimpanzees and other non-human primates were injected with expression vectors containing inserts for HIV-l gagipol and envlrev genes. Injections included the use of a non-peptidic facilitator, bupivacaine-HCl. Resulting immune responses were broad, individualized, and led to a decrease in viral load in HIV-l-infected primates3.5. In addition, naive vaccinated chimpanzees were protected against heterologous challenge4. The results of those experiments led to the initiation of phase I human clinical trials. Our initial results demonstrate the enhancement of humoral immune responses specific to HIV-l in infected patients after therapeutic inoculation with our nucleic acid vaccine constructs.
DNA vaccination
MATERIALS
AND METHODS
Use of human subjects The Clinical Research Center (CRC) at the Hospital of the University of Pennsylvania served as the site for human vaccinations. Three groups of five subjects each received three intramuscular vaccinations at IO-week intervals. The injected solution contained 30, 100, or 300 ,Llg of the nucleic acid construct (expressing gpl60MN = pCMN160)with a bupivacaine-HCI facilitator. The protocol was approved by the University of Pennsylvania’s Committee on Studies Involving Human Beings. Each subject provided informed consent for participation in the project. Throughout the study, the subjects received data about their progress and were reminded that they could withdraw from the project at any time. Patients were monitored during the course of the study by physical examination, and clinical laboratory parameters. Immunological parameters including antibody and cellular responses against gp120 were also evaluated. ELISA assays Human serum samples were prepared and analysed by standard ELISA techniques9, “. Briefly, 96-well Immulon II microtitre assay plates (Fisher Scientific Pittsburgh, PA) were coated with recombinant HIV-I MN gp120 (Immunodiagnostics Bedford, MA), or with a peptide corresponding to the V3 immunodominant of the gpl20MN envelope glycoprotein region (obtained from the AIDS Research and Reference Reagent Program). Sera samples were serial diluted in
0.500 0.475 0.450 0.425 0.400 0.375 0.350 0.325 0.300 0.275 0.250 0.225 0.200 0.175 0.150 0.125
with HIV-1 constructs:
K.E. Ugen et al.
dilution buffer (5% non-fat dry milk in PBS (pH 7.2) containing 0.05% Tween-20), then applied to the coated plates. Conjugated anti-human polyvalent immunoglobulin (Ig)-HRP (Sigma, St. Louis, MO) was used to determine binding of specific human antisera. Binding activity was measured at an optical density (A) of 450 nm on an Emax ELISA reader (Molecular Devices Sunnyvale, CA). Negative and non-specific binding controls were provided with pooled normal (seronegative) human sera and non-viral coating proteins/peptides. Specific binding &,, were calculated by subtracting AJS,, values for binding of antisera to BSA coated wells from those for binding of antisera to recombinant gp120 or V3 peptide (Figrre I). Calculations were also performed to express percent enhancement of binding to the gpl20 V3MN loop peptide after vaccination (F@tve 2). Statistical analysis Statistical differences between responses before and after vaccination were determined by a paired student t-test.
RESULTS AND DISCUSSION In this report we summarize some of the serological analysis on the human study group vaccinated with the 100 /lg dose of the DNA plasmid vaccine. The vaccine was administered according to the regimen described above in the Materials and Methods section. For the serological analysis sera was collected at time points
’ 1 : 1 : : : -
l prevaccinated q Wk 21 postvaccination q Wk 36 postvaccination
0.100 1 0.075 -
1:65,536
1: 131,072 Dilutions
1:262,144
1:524,288
of Sera
Figure 1 Enhancement of anti-gpl20MN antibody responses in HIV-l-infected humans after DNA plasmid vaccination. the mean Adsonmvalues versus sera dilutions for the 100 fig treatment group as described in the text
Vaccine
The results show
1998 Volume 16 Number 19
1819
DNA vaccination with HIV-7 constructs: K.E. Ugen et al.
Weeks Post-Vaccination
1:4,500
1: 13,500 Dilutions
of Sera
Figure 2 Enhancement of anti-V3 loop antibody responses in HIV-l-infected vaccine. The results show mean per cent enhancement of antibody response pre-vaccination responses. See text for further details
prior to (i.e. pre sample) the first inoculation with the envelope expressing construct and at time points after the first, second and third inoculations. ELISA analysis was performed to determine the level of anti-gpl20 antibody present in the sera samples before and after inoculation with the DNA plasmid vaccine. The sera dilution range utilized in this report was 1:65536 to 1:524288. The initial dilution in the series was relatively high because of the presence of pre-existing anti-gp120 antibodies due to HIV-l infection. Figure 2 illustrates binding levels of patient sera samples from the 100 pg treatment cohort to recombinant gpl20MN protein measured before and after vaccination. Binding A450nmvalues are shown for week 21 (i.e. 1 week after the second vaccination) and week 36 (i.e. 6 weeks after the third vaccination). The data indicate that binding of antisera from the subjects to gp120 is increased after vaccination. &,,“,,, values are significantly increased at both weeks 21 and 36 compared with the pre-vaccination value at dilutions of 1:65536, 1:131072 and 1:262 144. Significance is at the 95% confidence level by measurement through the paired t-test. In addition, binding of the pre- and postvaccination antisera were also measured against a V3 loop peptide derived from gpl20MN (aa 301-320, CTRPNYNKRKRIHIGPGRAF). Figure 2 summarizes the data from this analysis. The data in the figure are shown as the mean per cent enhancement of binding to V3 loop of antisera from the 100 pugvaccine cohort compared with the binding of antisera before vaccination. In addition, we wanted to evaluate antibody binding in a time
1820
Vaccine
1998 Volume 16 Number 19
humans after a single vaccination with a DNA plasmid at various intervals after a single vaccination relative to
course after a single vaccination with the construct. Therefore, measurements were made at 2, 4 and 8 weeks after a single 100 pg vaccine dose in this patient cohort. The data indicate a time-related increase in binding to gp120 after vaccination at both the 1:4500 and 1:13500 dilutions, i.e. a per cent enhancement of binding at the 1:4500 dilution of 9.84, 21 and 47.6 at weeks 2, 4 and 8, respectively. Likewise, the values at the 1:13500 dilution were 2.978, 4.208 and 35.57 at weeks 2, 4 and 8, respectively.
SUMMARY
AND CONCLUSIONS
This report summarizes some of the initial serological analysis of the HIV-l envelope glycoprotein-expressing DNA construct. The results of the phase I human clinical trial indicated that the vaccine preparation was well tolerated in the individuals, that is, no significant adverse clinical events were noted. In addition, a boosting of humoral immune responses was apparent in the individuals after vaccination. This boosting effect was noted against recombinant gp120 and a V3 loop peptide from gp120. Therefore, the DNA plasmid-based vaccine regimen apparently produces a biological effect in vaccinated individuals, as evidenced by a boosted humoral immune response. These data demonstrate the potential of the DNA plasmid vaccine approach in humans. Therefore, further studies establishing the potential efficacy of this technique are warranted.
DNA vaccination with HIV-1 constructs: K.E. Ugen et al. ACKNOWLEDGEMENTS This work was supported in part by grants to DBW from the National Institutes of Health including a SPIRAT grant. MLB was supported through an NIAID Mentored Clinical Scientist Development Award. KEU was supported by grants from the University of South Florida Research Foundation, the Oak Ridge Associatcd Universities and NIH (ROl-HL59818). We also acknowledge the AIDS Research Reference and Reagent Repository for supplying the HIV-I MN V.3 loop peptide.
4
5
6
7
a
REFERENCES Desrosiers, Ft. C. Safety issues facing development of a liveattenuated, multiply deleted HIV-1 vaccine. ADS Res. Human Retroviruses 1994, 10, 331-332. Kampinga, G. A., Simonon, A., Van De Perre, P., Karita, E., Msellati, P. and Goudsrnit, J. Findings of heterogeneity at seroconversion, coinfection, and recombinants of HIV-l subtypes A and C. Virology 1997, 227,63-76. Boyer, J. D., Ugen K. E., Chattergoon M., et al. DNA vaccination as anti-HIV immunotherapy in infected chimpanzees. J. Infect. Dis.. 1997, 176, 1501-1509.
9
10
Boyer, J. D., Ugen, K. E. and Wang, B. et al. Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination. Nature fvled. 1997, 3, 526532. Boyer, J. D., Wang, B. and Srikantan, V. et al. Induction of humoral and cellular immune responses to the human immunodeficiency type 1 virus in non-human primates by in viva DNA inoculation. Virology 1995, 211, 102-l 12. Ugen, K. E., Boyer, J. D. and Wang, B. et a/. Nucleic acid immunization of chimpanzees prophylactic/immunotherapeutic vaccination model for HIV-l: prelude to a clinical trial. Vaccine 1997, 15, 927-930. Wang, B., Boyer, J. and Srikantan, V. et a/. DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type 1 in mice and non-human primates. DNA Cell Biol. 1993, 12, 799-805. Wang, B., Ugen, K. E. and Srikantan, V. et al. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc. Nat/. Acad. Sci. U.S.A. 1993, 90, 4156-4160. Ugen, K. E., Goedert, J. J. and Boyer, J. et al. Vertical transmission of human immunodeficiency virus (HIV) infection. Reactivity of maternal sera with glycoprotein 120 and 41 peptides from HIV type 1. J. C/in. invest 1992, 89, 1923-1930. Ugen, K. E., Srikantan, V., Goedert, J. J., Nelson, Ft. P., Williams, W. V. and Weiner, D. B. Vertical transmission of immunodeficiency virus type 1: seroreactivity by maternal antibodies to the carboxy region of the gp41 envelope. J. Infect. Dis. 1997, 175, 63-69.
Vaccine 1998 Volume 16 Number 19
1821
Vaccine, Vol. 16, No. 19, pp. 1818-1821, 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410)(/98 $19+0.00
ELSEVIER
DNA vaccination with HIV-l expressing constructs elicits immune responses in humans Kenneth E. Ugen”7, Susan B. Nyland*, Jean D. Bayer?, Cristina Vidal*, Liana Lera”, Sowsan Rasheid”, Michael Chattergoon?, Mark L. Bagarazzil, Richard Ciccarelli§, Terry Higginss, Yaila Baines, Richard Ginsbergs, Rob Roy Macgregor? and David B. Weiner? Humoral and cellular immune responses have been produced by intramuscular vaccination with DNA plasmids expressing HIV1 genes, suggesting possible immunotherapeutic and prophylactic value for these constructs. Vaccination with these constructs has decreased HIV-1 viral load in HIV-l-infected chimpanzees. In addition, naive (i.e. non-HIVl-infected) chimpanzees were protected against a heterologous challenge with HIV-l. Ongoing phase I clinical trials show that therapeutic vaccinations indeed boost anti-HIV-I immune responses in humans. A therapeutic phase I trial on humans with these constructs induced a good safety profile and also demonstrated an immunological potentiation. These findings indicate that further studies with these constructs in humans are warranted. 0 1998 Elsevier Science Ltd. All rights reserved Keywords: Antibody; ELISA; DNA plasmid vaccination
The human immunodeficiency virus type I, HIV-l, continues to challenge researchers due to its ability to escape immune attack and to mutate into multidrug resistant strains. Therefore, the development of effective prophylactic and therapeutic strategies which emphasize enhancement of immunity without increasing the selection pressure for drug resistant mutants is sorely needed. The use of multidrug ‘cocktail’ therapies which include the newer protease inhibitors has reduced the viral load in many patients at the present time. The benefits of an immunoenhancing, non-mutagenic therapeutic system for these individuals might be best realized during the period when viral load is reduced and before multidrug resistant mutants become established. The prevention of future HIV-l infections in high risk populations would require vaccines capable of eliciting immune responses (most likely humoral and cell-mediated). The utility of live attenuated viral preparations against *University of South Florida College of Medicine, Department of Medical Microbiology and Immunology, 12901 Bruce 6. Downs Blvd, Tampa, FL 33612, USA. funiversity of Pennsylvania School of Medicine, Department of Pathology and Laboratory Medicine, 422 Curie Blvd, Room 505, Philadelphia, PA 19104, USA. *Allegheny University of the Health Sciences, Department of Pediatrics, St. Christopher’s Hospital for Children, Front Street and Erie Avenue, Philadelphia, PA 19134, USA. QApollon Inc., Malvern, PA 19355, USA. lITo whom correspondence should be addressed. Tel.: (813) 9741917; Fax: (813) 9744151; E-mail: kugen@coml .med.usf.edu
1818
Vaccine
1998 Volume
16 Number
19
HIV-l has been suggested’. Using this approach for the prevention of HIV-l infection has the obvious advantage of preparing both arms of the immune response for future viral challenge. However, the use of an attenuated HIV-l on high risk individuals who may or may not already be harbouring an undetectable level of virus leads to questions concerning recombination, reversion, and pathogenic expression of new recombinant?. Therefore, vaccine approaches which ‘mimic’ live attenuated preparations without the associated safety concerns are needed. We previously reported on the use of direct nucleic acid inoculation as a novel alternative to live attenuated virus3-‘. This system appears to mimic natural infection in that it may enable potential antigen presenting cells (APC) to process antigenic peptides through MHC I and MHC II pathways, yet is non-infectious. Chimpanzees and other non-human primates were injected with expression vectors containing inserts for HIV-l gagipol and envlrev genes. Injections included the use of a non-peptidic facilitator, bupivacaine-HCl. Resulting immune responses were broad, individualized, and led to a decrease in viral load in HIV-l-infected primates3.5. In addition, naive vaccinated chimpanzees were protected against heterologous challenge4. The results of those experiments led to the initiation of phase I human clinical trials. Our initial results demonstrate the enhancement of humoral immune responses specific to HIV-l in infected patients after therapeutic inoculation with our nucleic acid vaccine constructs.
DNA vaccination
MATERIALS
AND METHODS
Use of human subjects The Clinical Research Center (CRC) at the Hospital of the University of Pennsylvania served as the site for human vaccinations. Three groups of five subjects each received three intramuscular vaccinations at IO-week intervals. The injected solution contained 30, 100, or 300 ,Llg of the nucleic acid construct (expressing gpl60MN = pCMN160)with a bupivacaine-HCI facilitator. The protocol was approved by the University of Pennsylvania’s Committee on Studies Involving Human Beings. Each subject provided informed consent for participation in the project. Throughout the study, the subjects received data about their progress and were reminded that they could withdraw from the project at any time. Patients were monitored during the course of the study by physical examination, and clinical laboratory parameters. Immunological parameters including antibody and cellular responses against gp120 were also evaluated. ELISA assays Human serum samples were prepared and analysed by standard ELISA techniques9, “. Briefly, 96-well Immulon II microtitre assay plates (Fisher Scientific Pittsburgh, PA) were coated with recombinant HIV-I MN gp120 (Immunodiagnostics Bedford, MA), or with a peptide corresponding to the V3 immunodominant of the gpl20MN envelope glycoprotein region (obtained from the AIDS Research and Reference Reagent Program). Sera samples were serial diluted in
0.500 0.475 0.450 0.425 0.400 0.375 0.350 0.325 0.300 0.275 0.250 0.225 0.200 0.175 0.150 0.125
with HIV-1 constructs:
K.E. Ugen et al.
dilution buffer (5% non-fat dry milk in PBS (pH 7.2) containing 0.05% Tween-20), then applied to the coated plates. Conjugated anti-human polyvalent immunoglobulin (Ig)-HRP (Sigma, St. Louis, MO) was used to determine binding of specific human antisera. Binding activity was measured at an optical density (A) of 450 nm on an Emax ELISA reader (Molecular Devices Sunnyvale, CA). Negative and non-specific binding controls were provided with pooled normal (seronegative) human sera and non-viral coating proteins/peptides. Specific binding &,, were calculated by subtracting AJS,, values for binding of antisera to BSA coated wells from those for binding of antisera to recombinant gp120 or V3 peptide (Figrre I). Calculations were also performed to express percent enhancement of binding to the gpl20 V3MN loop peptide after vaccination (F@tve 2). Statistical analysis Statistical differences between responses before and after vaccination were determined by a paired student t-test.
RESULTS AND DISCUSSION In this report we summarize some of the serological analysis on the human study group vaccinated with the 100 /lg dose of the DNA plasmid vaccine. The vaccine was administered according to the regimen described above in the Materials and Methods section. For the serological analysis sera was collected at time points
’ 1 : 1 : : : -
l prevaccinated q Wk 21 postvaccination q Wk 36 postvaccination
0.100 1 0.075 -
1:65,536
1: 131,072 Dilutions
1:262,144
1:524,288
of Sera
Figure 1 Enhancement of anti-gpl20MN antibody responses in HIV-l-infected humans after DNA plasmid vaccination. the mean Adsonmvalues versus sera dilutions for the 100 fig treatment group as described in the text
Vaccine
The results show
1998 Volume 16 Number 19
1819
DNA vaccination with HIV-7 constructs: K.E. Ugen et al.
Weeks Post-Vaccination
1:4,500
1: 13,500 Dilutions
of Sera
Figure 2 Enhancement of anti-V3 loop antibody responses in HIV-l-infected vaccine. The results show mean per cent enhancement of antibody response pre-vaccination responses. See text for further details
prior to (i.e. pre sample) the first inoculation with the envelope expressing construct and at time points after the first, second and third inoculations. ELISA analysis was performed to determine the level of anti-gpl20 antibody present in the sera samples before and after inoculation with the DNA plasmid vaccine. The sera dilution range utilized in this report was 1:65536 to 1:524288. The initial dilution in the series was relatively high because of the presence of pre-existing anti-gp120 antibodies due to HIV-l infection. Figure 2 illustrates binding levels of patient sera samples from the 100 pg treatment cohort to recombinant gpl20MN protein measured before and after vaccination. Binding A450nmvalues are shown for week 21 (i.e. 1 week after the second vaccination) and week 36 (i.e. 6 weeks after the third vaccination). The data indicate that binding of antisera from the subjects to gp120 is increased after vaccination. &,,“,,, values are significantly increased at both weeks 21 and 36 compared with the pre-vaccination value at dilutions of 1:65536, 1:131072 and 1:262 144. Significance is at the 95% confidence level by measurement through the paired t-test. In addition, binding of the pre- and postvaccination antisera were also measured against a V3 loop peptide derived from gpl20MN (aa 301-320, CTRPNYNKRKRIHIGPGRAF). Figure 2 summarizes the data from this analysis. The data in the figure are shown as the mean per cent enhancement of binding to V3 loop of antisera from the 100 pugvaccine cohort compared with the binding of antisera before vaccination. In addition, we wanted to evaluate antibody binding in a time
1820
Vaccine
1998 Volume 16 Number 19
humans after a single vaccination with a DNA plasmid at various intervals after a single vaccination relative to
course after a single vaccination with the construct. Therefore, measurements were made at 2, 4 and 8 weeks after a single 100 pg vaccine dose in this patient cohort. The data indicate a time-related increase in binding to gp120 after vaccination at both the 1:4500 and 1:13500 dilutions, i.e. a per cent enhancement of binding at the 1:4500 dilution of 9.84, 21 and 47.6 at weeks 2, 4 and 8, respectively. Likewise, the values at the 1:13500 dilution were 2.978, 4.208 and 35.57 at weeks 2, 4 and 8, respectively.
SUMMARY
AND CONCLUSIONS
This report summarizes some of the initial serological analysis of the HIV-l envelope glycoprotein-expressing DNA construct. The results of the phase I human clinical trial indicated that the vaccine preparation was well tolerated in the individuals, that is, no significant adverse clinical events were noted. In addition, a boosting of humoral immune responses was apparent in the individuals after vaccination. This boosting effect was noted against recombinant gp120 and a V3 loop peptide from gp120. Therefore, the DNA plasmid-based vaccine regimen apparently produces a biological effect in vaccinated individuals, as evidenced by a boosted humoral immune response. These data demonstrate the potential of the DNA plasmid vaccine approach in humans. Therefore, further studies establishing the potential efficacy of this technique are warranted.
DNA vaccination with HIV-1 constructs: K.E. Ugen et al. ACKNOWLEDGEMENTS This work was supported in part by grants to DBW from the National Institutes of Health including a SPIRAT grant. MLB was supported through an NIAID Mentored Clinical Scientist Development Award. KEU was supported by grants from the University of South Florida Research Foundation, the Oak Ridge Associatcd Universities and NIH (ROl-HL59818). We also acknowledge the AIDS Research Reference and Reagent Repository for supplying the HIV-I MN V.3 loop peptide.
4
5
6
7
a
REFERENCES Desrosiers, Ft. C. Safety issues facing development of a liveattenuated, multiply deleted HIV-1 vaccine. ADS Res. Human Retroviruses 1994, 10, 331-332. Kampinga, G. A., Simonon, A., Van De Perre, P., Karita, E., Msellati, P. and Goudsrnit, J. Findings of heterogeneity at seroconversion, coinfection, and recombinants of HIV-l subtypes A and C. Virology 1997, 227,63-76. Boyer, J. D., Ugen K. E., Chattergoon M., et al. DNA vaccination as anti-HIV immunotherapy in infected chimpanzees. J. Infect. Dis.. 1997, 176, 1501-1509.
9
10
Boyer, J. D., Ugen, K. E. and Wang, B. et al. Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination. Nature fvled. 1997, 3, 526532. Boyer, J. D., Wang, B. and Srikantan, V. et al. Induction of humoral and cellular immune responses to the human immunodeficiency type 1 virus in non-human primates by in viva DNA inoculation. Virology 1995, 211, 102-l 12. Ugen, K. E., Boyer, J. D. and Wang, B. et a/. Nucleic acid immunization of chimpanzees prophylactic/immunotherapeutic vaccination model for HIV-l: prelude to a clinical trial. Vaccine 1997, 15, 927-930. Wang, B., Boyer, J. and Srikantan, V. et a/. DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type 1 in mice and non-human primates. DNA Cell Biol. 1993, 12, 799-805. Wang, B., Ugen, K. E. and Srikantan, V. et al. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc. Nat/. Acad. Sci. U.S.A. 1993, 90, 4156-4160. Ugen, K. E., Goedert, J. J. and Boyer, J. et al. Vertical transmission of human immunodeficiency virus (HIV) infection. Reactivity of maternal sera with glycoprotein 120 and 41 peptides from HIV type 1. J. C/in. invest 1992, 89, 1923-1930. Ugen, K. E., Srikantan, V., Goedert, J. J., Nelson, Ft. P., Williams, W. V. and Weiner, D. B. Vertical transmission of immunodeficiency virus type 1: seroreactivity by maternal antibodies to the carboxy region of the gp41 envelope. J. Infect. Dis. 1997, 175, 63-69.
Vaccine 1998 Volume 16 Number 19
1821