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1.
Biochemistry ; 42(11): 3214-23, 2003 Mar 25.
Article in English | MEDLINE | ID: mdl-12641452

ABSTRACT

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.


Subject(s)
AIDS Vaccines/immunology , Antibodies, Monoclonal/immunology , HIV Envelope Protein gp41/immunology , HIV-1/immunology , AIDS Vaccines/chemistry , Amino Acid Sequence , Enzyme-Linked Immunosorbent Assay , HIV Envelope Protein gp41/chemistry , Models, Molecular , Molecular Sequence Data , Structure-Activity Relationship
2.
Protein Expr Purif ; 23(2): 270-81, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11676602

ABSTRACT

A recently developed method for the identification and quantitation of antigen-specific T lymphocytes involves the use of complexes of biotinylated major histocompatibility complex (MHC) and avidin conjugated to a fluorescent reporter group. This complex, dubbed the "tetramer," binds to antigen-specific T lymphocytes in vitro, which can then be sorted and counted by fluorescence-activated flow cytometry to measure immune response. Our research has focused on developing the purification process for preparing tetramer reagent. Our goal was to reengineer a published lab-scale purification process to reduce the number of processing steps and to make the process scalable. In our reengineered process, recombinant MHC alpha chain is isolated from Escherichia coli as inclusion bodies by tangential flow filtration. The purified MHC alpha chain is refolded with beta-2-microglobulin and the target peptide antigen to form the class I MHC. The resulting MHC is purified by hydrophobic interaction chromatography (HIC) and biotinylated enzymatically, and the biotinylated MHC is purified by a second HIC step. The tetramer is prepared by mixing biotinylated MHC with an avidin-fluorophore conjugate. The tetramer is further purified to remove any excess MHC or avidin components. Analysis by flow cytometry confirmed that the tetramers generated by this new process gave bright staining and specific binding to CD3+/CD8+ cells of vaccinated monkeys and led to results that were equivalent to those generated with tetramer produced by the original process.


Subject(s)
Gene Products, gag/immunology , Histocompatibility Antigens Class I/isolation & purification , Simian Immunodeficiency Virus/immunology , T-Lymphocytes/immunology , Animals , Biotin/chemistry , Biotin/isolation & purification , Biotinylation , Cell Separation/methods , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Flow Cytometry , Fluorescent Dyes/chemistry , Fluorescent Dyes/isolation & purification , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/immunology , Inclusion Bodies , Macaca mulatta , Macromolecular Substances , Major Histocompatibility Complex , Protein Folding , Recombinant Proteins/isolation & purification , SAIDS Vaccines/administration & dosage , SAIDS Vaccines/immunology , beta 2-Microglobulin/chemistry , beta 2-Microglobulin/immunology , beta 2-Microglobulin/isolation & purification
3.
Immunol Lett ; 79(1-2): 57-61, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11595290

ABSTRACT

Accumulating evidence has demonstrated the importance of cytotoxic T lymphocytes (CTLs) and helper T lymphocytes in controlling HIV-1 replication. We have elicited immune responses in rhesus monkeys utilizing DNA vaccines augmented by the administration of IL-2/Ig, a fusion protein consisting of interleukin-2 and the Fc portion of IgG2. These vaccine-elicited immune responses did not prevent infection following a high-dose intravenous challenge with SHIV(89.6P) but did control viremia to nearly undetectable levels and prevented immunodeficiency and clinical disease. In contrast, control monkeys developed high levels of viremia and exhibited a rapid loss of CD4(+) T cells, significant clinical disease progression, and death in half of the animals by day 140 following challenge. Vaccine approaches that elicit immune responses capable of reducing plasma viral loads, but not capable of inducing sterilizing immunity, may still provide substantial clinical benefits.


Subject(s)
AIDS Vaccines/pharmacology , Acquired Immunodeficiency Syndrome/immunology , Acquired Immunodeficiency Syndrome/prevention & control , Macaca mulatta/immunology , SAIDS Vaccines/pharmacology , Vaccines, DNA/pharmacology , Animals , CD4 Lymphocyte Count , HIV Antibodies/biosynthesis , HIV Infections/immunology , Immunoglobulin Fc Fragments/administration & dosage , Interleukin-2/administration & dosage , Neutralization Tests , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Viremia/immunology , Viremia/prevention & control
4.
J Virol ; 75(5): 2462-7, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11160750

ABSTRACT

Increasing evidence suggests that the generation of cytotoxic T-lymphocyte (CTL) responses specific for a diversity of viral epitopes will be needed for an effective human immunodeficiency virus type 1 (HIV-1) vaccine. Here, we determine the frequencies of CTL responses specific for the simian immunodeficiency virus Gag p11C and HIV-1 Env p41A epitopes in simian-human immunodeficiency virus (SHIV)-infected and vaccinated rhesus monkeys. The p11C-specific CTL response was high frequency and dominant and the p41A-specific CTL response was low frequency and subdominant in both SHIV-infected monkeys and in monkeys vaccinated with recombinant modified vaccinia virus Ankara vectors expressing these viral antigens. Interestingly, we found that plasmid DNA vaccination led to high-frequency CTL responses specific for both of these epitopes. These data demonstrate that plasmid DNA may be useful in eliciting a broad CTL response against multiple epitopes.


Subject(s)
AIDS Vaccines/immunology , Epitopes, T-Lymphocyte/immunology , SAIDS Vaccines/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccines, DNA/immunology , Animals , Gene Products, gag/genetics , Gene Products, gag/immunology , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/immunology , HIV Infections/immunology , HIV Infections/prevention & control , HIV-1/immunology , Humans , Immunodominant Epitopes/immunology , Macaca mulatta , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Immunodeficiency Virus/immunology , Vaccination
5.
J Virol ; 75(1): 73-82, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11119575

ABSTRACT

Cytotoxic T-lymphocyte (CTL) responses have been implicated as playing an important role in control of human immunodeficiency virus (HIV) infection. However, it is technically difficult to demonstrate CTL responses consistently in nonhuman primate and human subjects using traditional cytotoxicity assay methods. In this study, we systematically evaluated culture conditions that may affect the proliferation and expansion of CTL effector cells and presented a sensitive method for detection of cytotoxicity responses with bulk CTL cultures. We confirmed the sensitivity and specificity of this method by demonstration of vigorous CTL responses in a simian-HIV (SHIV)-infected rhesus macaque. The expansion of epitope-specific CTL effector cells was also measured quantitatively by CTL epitope-major histocompatibility complex tetramer complex staining. In addition, two new T-cell determinants in the SIV gag region are identified. Last, we showed the utility of this method for studying CTL responses in chimpanzee and human subjects.


Subject(s)
Acquired Immunodeficiency Syndrome/immunology , HIV-1 , Simian Acquired Immunodeficiency Syndrome/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Humans , Interleukin-7/pharmacology , Macaca mulatta
6.
Science ; 290(5491): 486-92, 2000 Oct 20.
Article in English | MEDLINE | ID: mdl-11039923

ABSTRACT

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.


Subject(s)
AIDS Vaccines/therapeutic use , Acquired Immunodeficiency Syndrome/prevention & control , HIV Infections/therapy , HIV-1 , Interleukin-2/therapeutic use , Vaccines, DNA/therapeutic use , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , Disease Progression , HIV Antibodies/blood , HIV Antibodies/immunology , HIV Infections/immunology , HIV Infections/virology , HIV-1/genetics , HIV-1/immunology , HIV-1/physiology , Humans , Interleukin-2/genetics , Interleukin-2/immunology , Lymphocyte Activation , Macaca mulatta , Neutralization Tests , Recombinant Fusion Proteins/therapeutic use , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Acquired Immunodeficiency Syndrome/therapy , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/immunology , Simian Immunodeficiency Virus/physiology , T-Lymphocytes, Cytotoxic/immunology , Vaccination , Viral Load , Viremia , Virus Replication
7.
J Virol ; 74(16): 7485-95, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10906202

ABSTRACT

The potential contribution of a plasmid DNA construct to vaccine-elicited protective immunity was explored in the simian immunodeficiency virus (SIV)/macaque model of AIDS. Making use of soluble major histocompatibility class I/peptide tetramers and peptide-specific killing assays to monitor CD8(+) T-lymphocyte responses to a dominant SIV Gag epitope in genetically selected rhesus monkeys, a codon-optimized SIV gag DNA vaccine construct was shown to elicit a high-frequency SIV-specific cytotoxic T-lymphocyte (CTL) response. This CTL response was demonstrable in both peripheral blood and lymph node lymphocytes. Following an intravenous challenge with the highly pathogenic viral isolate SIVsm E660, these vaccinated monkeys developed a secondary CTL response that arose with more rapid kinetics and reached a higher frequency than did the postchallenge CTL response in control plasmid-vaccinated monkeys. While peak plasma SIV RNA levels were comparable in the experimentally and control-vaccinated monkeys during the period of primary infection, the gag plasmid DNA-vaccinated monkeys demonstrated better containment of viral replication by 50 days following SIV challenge. These findings indicate that a plasmid DNA vaccine can elicit SIV-specific CTL responses in rhesus monkeys, and this vaccine-elicited immunity can facilitate the generation of secondary CTL responses and control of viral replication following a pathogenic SIV challenge. These observations suggest that plasmid DNA may prove a useful component of a human immunodeficiency virus type 1 vaccine.


Subject(s)
Gene Products, gag/immunology , SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccines, DNA/immunology , Animals , CD4 Lymphocyte Count , Gene Products, gag/genetics , Gene Products, gag/metabolism , Humans , Lymph Nodes/immunology , Lymph Nodes/virology , Macaca mulatta , RNA, Viral/analysis , SAIDS Vaccines/administration & dosage , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/physiology , Vaccination , Vaccines, DNA/administration & dosage , Viral Load , Virus Replication
8.
Proc Natl Acad Sci U S A ; 97(8): 4192-7, 2000 Apr 11.
Article in English | MEDLINE | ID: mdl-10759543

ABSTRACT

The potential utility of plasmid DNA as an HIV-1 vaccination modality currently is an area of active investigation. However, recent studies have raised doubts as to whether plasmid DNA alone will elicit immune responses of sufficient magnitude to protect against pathogenic AIDS virus challenges. We therefore investigated whether DNA vaccine-elicited immune responses in rhesus monkeys could be augmented by using either an IL-2/Ig fusion protein or a plasmid expressing IL-2/Ig. Sixteen monkeys, divided into four experimental groups, were immunized with (i) sham plasmid, (ii) HIV-1 Env 89.6P and simian immunodeficiency virus mac239 Gag DNA vaccines alone, (iii) these DNA vaccines and IL-2/Ig protein, or (iv) these DNA vaccines and IL-2/Ig plasmid. The administration of both IL-2/Ig protein and IL-2/Ig plasmid induced a significant and sustained in vivo activation of peripheral T cells in the vaccinated monkeys. The monkeys that received IL-2/Ig plasmid generated 30-fold higher Env-specific antibody titers and 5-fold higher Gag-specific, tetramer-positive CD8+ T cell levels than the monkeys receiving the DNA vaccines alone. IL-2/Ig protein also augmented the vaccine-elicited immune responses, but less effectively than IL-2/Ig plasmid. Augmentation of the immune responses by IL-2/Ig was evident after the primary immunization and increased with subsequent boost immunizations. These results demonstrate that the administration of IL-2/Ig plasmid can substantially augment vaccine-elicited humoral and cellular immune responses in higher primates.


Subject(s)
HIV-1/immunology , Immunoglobulin G/administration & dosage , Interleukin-2/administration & dosage , Simian Immunodeficiency Virus/immunology , Vaccines, DNA/immunology , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Gene Products, env/immunology , HIV Antibodies/biosynthesis , HIV-1/genetics , Macaca mulatta , Plasmids/administration & dosage , Receptors, Interleukin-2/immunology , Simian Immunodeficiency Virus/genetics , T-Lymphocytes, Cytotoxic/immunology
9.
Vaccine ; 17(22): 2862-72, 1999 Jul 16.
Article in English | MEDLINE | ID: mdl-10438057

ABSTRACT

We report on the properties of a set of HIV-1 IIIB Env mutants carrying a linear gp41 epitope insertion (LLELDKWASL) in the V1, V2, V3 or V4 variable loop. Insertion of the epitope, which is defined by the HIV-1 neutralizing MAb 2F5, was well tolerated in the V1, V2 and V4 loops, as these mutants were properly expressed, retained reactivity to conformation-dependent monoclonal antibodies and exhibited patterns similar to the parental Env molecule. However, insertion of this epitope in the V3 loop was associated with drastically reduced protein expression. Relative to parental Env molecule, the V1, V2 and V4 insertion mutants demonstrated significantly increased binding to mAb 2F5 in vitro. To evaluate immunogenicity, mice and guinea pigs were immunized with plasmid expression vectors for the mutant proteins. For both mice and guinea pigs, all four mutants elicited anti-gp120 antibody responses. In mice the V1 and V3 insertion mutants, but neither the V2 or V4 insertion mutant nor the parental env, elicited significant titers against the epitope peptide, whereas in guinea pigs, V2 insertion mutant was most effective in eliciting anti-2F5 peptide antibody responses. While original V2 2F5 insertion mutant failed to elicit anti-2F5 peptide responses in mice, studies with 14 additional V2 insertion mutants revealed several insertion sites at which the epitope was able to induce epitope-specific antibody responses. This indicates that the precise position at which the epitope insertion takes place dictates the ability of the mutant to induce the epitope-specific antibody responses. When tested for virus neutralization activity, the guinea pig sera that contain high titers of anti-2F5 peptide antibody failed to enhance the virus neutralizing activity, suggesting that the configuration of 2F5 epitope plays a critical role in inducing neutralizing antibody responses. The results from this study may have potential implications with respect to modification of the HIV-1 Env molecule for the purpose of improving HIV-1 Env immunogenicity.


Subject(s)
AIDS Vaccines/genetics , AIDS Vaccines/immunology , Epitopes/genetics , Epitopes/immunology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , Vaccines, Synthetic/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Viral/biosynthesis , Cell Line , Female , Guinea Pigs , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutagenesis, Insertional , Neutralization Tests , Vaccines, Synthetic/genetics
10.
J Immunol ; 161(4): 1875-82, 1998 Aug 15.
Article in English | MEDLINE | ID: mdl-9712056

ABSTRACT

The use of cytokines has shown promise as an approach for amplifying vaccine-elicited immune responses, but the application of these immunomodulatory molecules in this setting has not been systematically explored. In this report we investigate the use of protein- and plasmid-based cytokines to augment immune responses elicited by an HIV-1 gp120 plasmid DNA vaccine (pV1J-gp120) in mice. We demonstrate that immune responses elicited by pV1J-gp120 can be either augmented or suppressed by administration of plasmid cytokines. A dicistronic plasmid expressing both gp120 and IL-2 induced a surprisingly weaker gp120-specific immune response than did the monocistronic pV1J-gp120 plasmid. In contrast, systemic delivery of soluble IL-2/Ig fusion protein following pV1J-gp120 vaccination significantly amplified the gp120-specific immune response as measured by Ab, proliferative, and CTL levels. Administration of plasmid IL-2/Ig had different effects on the DNA vaccine-elicited immune response that depended on the temporal relationship between Ag and cytokine delivery. Injection of plasmid IL-2/Ig either before or coincident with pV1J-gp120 suppressed the gp120-specific immune response, whereas injection of plasmid IL-2/Ig after pV1J-gp120 amplified this immune response. To maximize immune responses elicited by a DNA vaccine, therefore, it appears that the immune system should first be primed with a specific Ag and then amplified with cytokines. The data also show that IL-2/Ig is more effective than native IL-2 as a DNA vaccine adjuvant.


Subject(s)
AIDS Vaccines/immunology , Adjuvants, Immunologic/administration & dosage , Cytokines/genetics , HIV-1/immunology , Immunoglobulins/genetics , Immunosuppressive Agents/administration & dosage , Plasmids/immunology , Vaccines, DNA/immunology , AIDS Vaccines/administration & dosage , Adjuvants, Immunologic/physiology , Amino Acid Sequence , Animals , Antibodies, Viral/biosynthesis , Cytokines/administration & dosage , Cytokines/biosynthesis , Female , HIV Envelope Protein gp120/biosynthesis , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , Immunoglobulins/administration & dosage , Immunoglobulins/immunology , Immunosuppressive Agents/pharmacology , Injections, Intramuscular , Interleukin-2/administration & dosage , Interleukin-2/biosynthesis , Interleukin-2/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Molecular Sequence Data , Plasmids/administration & dosage , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/immunology , Solubility , Vaccines, DNA/administration & dosage
11.
Proc Natl Acad Sci U S A ; 94(17): 9378-83, 1997 Aug 19.
Article in English | MEDLINE | ID: mdl-9256490

ABSTRACT

It is generally thought that an effective vaccine to prevent HIV-1 infection should elicit both strong neutralizing antibody and cytotoxic T lymphocyte responses. We recently demonstrated that potent, boostable, long-lived HIV-1 envelope (Env)-specific cytotoxic T lymphocyte responses can be elicited in rhesus monkeys using plasmid-encoded HIV-1 env DNA as the immunogen. In the present study, we show that the addition of HIV-1 Env protein to this regimen as a boosting immunogen generates a high titer neutralizing antibody response in this nonhuman primate species. Moreover, we demonstrate in a pilot study that immunization with HIV-1 env DNA (multiple doses) followed by a final immunization with HIV-1 env DNA plus HIV-1 Env protein (env gene from HXBc2 clone of HIV IIIB; Env protein from parental HIV IIIB) completely protects monkeys from infection after i.v. challenge with a chimeric virus expressing HIV-1 env (HXBc2) on a simian immmunodeficiency virusmac backbone (SHIV-HXBc2). The potent immunity and protection seen in these pilot experiments suggest that a DNA prime/DNA plus protein boost regimen warrants active investigation as a vaccine strategy to prevent HIV-1 infection.


Subject(s)
AIDS Vaccines , DNA, Viral/administration & dosage , Gene Products, env/immunology , Genes, env , HIV Infections/prevention & control , HIV-1/immunology , Animals , Cytotoxicity, Immunologic , DNA, Viral/immunology , HIV Infections/immunology , Haplorhini , Humans , Immunization , Reassortant Viruses/immunology , T-Lymphocytes/immunology
12.
Vaccine ; 15(8): 884-7, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9234539

ABSTRACT

Plasmid DNA vaccines encoding HIV-1 env were used to immunize mice and nonhuman primates. Plasmids were prepared that produced either secreted gp120 or full-length gp160. Mice immunized with gp120 DNA developed strong antigen-specific antibody responses, CD8+ cytotoxic T lymphocytes (CTL) (following in vitro restimulation with gp120-derived peptide), and showed in vitro proliferation and Th1-like cytokine secretion [gamma-interferon, interleukin (IL)-2 with little or no IL-4] by lymphocytes obtained from all lymphatic compartments tested (spleen, blood, and inguinal, iliac, and mesenteric lymph nodes). This indicated that systemic anti-gp120 cell-mediated immunity was induced by this DNA vaccine. Although similar antibody responses were observed in mice immunized by either intramuscular or intradermal routes, T cell responses were significantly stronger in mice injected intramuscularly. Rhesus monkeys immunized with both gp120 and gp160 DNAs exhibited significant CD8+ CTL responses, following in vitro restimulation of peripheral blood lymphocytes with antigen. These experiments demonstrate that DNA immunization elicits potent immune responses against HIV env in both a rodent and a nonhuman primate species.


Subject(s)
AIDS Vaccines/immunology , Gene Products, env/immunology , HIV Antibodies/biosynthesis , HIV Envelope Protein gp120/immunology , HIV-1/genetics , Vaccines, DNA/immunology , AIDS Vaccines/administration & dosage , AIDS Vaccines/genetics , Animals , DNA, Viral/immunology , Gene Products, env/genetics , HIV Antibodies/immunology , HIV Envelope Protein gp120/administration & dosage , HIV Envelope Protein gp120/genetics , Macaca mulatta , Mice , Mice, Inbred BALB C , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
13.
Vaccine ; 15(8): 909-12, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9234545

ABSTRACT

The pre-clinical efficacy of DNA vaccines has been demonstrated in a number of animal models, but more limited data exist regarding their immunogenicity in non-human primates. The studies described below demonstrate that DNA vaccines in reasonable dosages encoding a variety of viral proteins could result in the generation of antibodies, neutralizing antibodies, or cytotoxic T lymphocytes in primates. Furthermore, these responses could be boosted by repeat administration of the DNA vaccine. In an effort to assess the safety of such vaccines sera from primates was shown to lack anti-DNA antibodies.


Subject(s)
RNA-Binding Proteins , Vaccines, DNA/administration & dosage , Animals , Antibodies, Viral/biosynthesis , Chlorocebus aethiops , DNA, Viral/immunology , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A virus/genetics , Influenza A virus/immunology , Influenza Vaccines/administration & dosage , Influenza Vaccines/immunology , Macaca mulatta , Male , Nucleocapsid Proteins , Nucleoproteins/genetics , Nucleoproteins/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccination , Vaccines, DNA/immunology , Viral Core Proteins/genetics , Viral Core Proteins/immunology
14.
J Immunol ; 158(9): 4471-7, 1997 May 01.
Article in English | MEDLINE | ID: mdl-9127013

ABSTRACT

The Th cell response elicited by an HIV-1 env plasmid DNA vaccine was assessed in rhesus monkeys by isolation of gp120-specific, MHC class II-restricted CD4+ T cell lines from PBL of vaccinated animals. The Env-specific CD4+ T cell lines recognized epitopes located in the second hypervariable region and in the carboxyl terminus of HIV-1 gp120. These cell lines proliferated in response to APC in the presence of recombinant gp120, as well as to APC expressing virally encoded Env. All of the CD4+ T cell lines responded to Env peptide by secreting IFN-gamma and TNF-alpha without appreciable IL-4 production. Recombinant gp120 stimulation of PBL from these vaccinated monkeys elicited the secretion of a similar profile of cytokines. Demonstration of a nucleotide vaccine eliciting a Th1-like immune response is consistent with the well documented ability of naked DNA vaccines to induce durable CD8+ CTL responses.


Subject(s)
AIDS Vaccines/immunology , CD4-Positive T-Lymphocytes/immunology , DNA, Viral/immunology , HIV-1/immunology , T-Lymphocytes, Helper-Inducer/immunology , Amino Acid Sequence , Animals , Epitope Mapping , Gene Products, env/immunology , HIV Envelope Protein gp120/immunology , Histocompatibility Antigens Class II/immunology , Interferon-gamma/metabolism , Lymphocyte Activation , Macaca mulatta , Molecular Sequence Data , Peptides/immunology , Tumor Necrosis Factor-alpha/metabolism
15.
Annu Rev Immunol ; 15: 617-48, 1997.
Article in English | MEDLINE | ID: mdl-9143702

ABSTRACT

Observations in the early 1990s that plasmid DNA could directly transfect animal cells in vivo sparked exploration of the use of DNA plasmids to induce immune responses by direct injection into animals of DNA encoding antigenic proteins. This method, termed DNA immunization, now has been used to elicit protective antibody and cell-mediated immune responses in a wide variety of preclinical animal models for viral, bacterial, and parasitic diseases. DNA vaccination is particularly useful for the induction of cytotoxic T cells. This review summarizes current knowledge on the vectors, immune responses, immunological mechanisms, safety considerations, and potential for further application of this novel method of immunization.


Subject(s)
Vaccines, DNA/pharmacology , Animals , Antibodies, Antinuclear/biosynthesis , Antibody Formation , Antigen Presentation , Autoimmunity , Bacterial Infections/immunology , Bacterial Infections/prevention & control , Disease Models, Animal , Genetic Vectors , Humans , Immune Tolerance , Immunity, Cellular , Immunization , Neoplasms, Experimental/immunology , Neoplasms, Experimental/prevention & control , Parasitic Diseases/immunology , Parasitic Diseases/prevention & control , Plasmids/genetics , Safety , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccines, DNA/adverse effects , Vaccines, DNA/genetics , Virus Diseases/immunology , Virus Diseases/prevention & control
16.
J Pharm Sci ; 85(12): 1317-24, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8961146

ABSTRACT

Recently it has been shown that immunization with plasmid DNA encoding genes for viral or bacterial antigens can elicit both humoral and cellular immune responses in rodents and nonhuman primates. In this study, mice and nonhuman primates were vaccinated by intramuscular injection with plasmids that express either a secreted form of HIV-1 gp120 or rev proteins. Mice receiving the tPA-gp120 DNA developed antigen-specific antibody responses against recombinant gp120 protein and the V2 peptide neutralization epitope as determined by ELISA. Vaccinated mice also exhibited gp120-specific T cell responses, such as in vitro proliferation of splenocytes and MHC Class I-restricted cytotoxic T lymphocyte (CTL) activities, following antigen restimulation. In addition, supernatants from these lymphocyte cultures showed high levels of gamma-interferon production compared with IL-4, suggesting that primarily type 1-like helper T (Th1) lymphocyte responses were induced by both vaccines. Th1-like responses were also obtained for mice vaccinated with rev DNA. Immune responses induced by gp120 or rev vaccines were dose-dependent, boostable, and long-lived (> or = 6 months). Nonhuman primates vaccinated with tPA-gp120 DNA also showed antigen-specific T lymphocyte proliferative and humoral responses, including moderate levels of neutralizing sera against homologous HIV. These results suggest that plasmid DNA may provide a powerful means for eliciting humoral and cellular immune responses against HIV.


Subject(s)
AIDS Vaccines/immunology , HIV-1/immunology , Vaccines, DNA/immunology , Amino Acid Sequence , Animals , Chlorocebus aethiops , Female , HIV Antibodies/blood , HIV Envelope Protein gp120/immunology , Lymphocyte Activation , Macaca mulatta , Mice , Mice, Inbred BALB C , Molecular Sequence Data
18.
Ann N Y Acad Sci ; 772: 198-208, 1995 Nov 27.
Article in English | MEDLINE | ID: mdl-8546393

ABSTRACT

Expression vectors encoding either HIV-1 gp160/rev, gp120, or rev alone were used for direct vaccination of mice and nonhuman primates. Each vaccine elicited long-lived (> 7 months) helper T cell responses in mice and monkeys as measured by in vitro proliferation of splenocytes following recombinant antigen treatment. Cytokine assays of the cell supernatants showed that approximately 100-fold more gamma-interferon than IL-4 was secreted during culture indicating that these vaccines elicited TH1-like responses. CD8+ CTL activities were also observed both in mice and rhesus. The gp120 and gp160/rev vaccines elicited antigen-specific antibodies, although these responses were more variable and lower magnitude for gp160/rev, and gp120 DNA-vaccinated African green monkeys had moderate levels of neutralizing antibodies. No antibodies were found against rev (an intracellular protein) with either rev vaccine. Similar antibody titers were obtained for gp120 by either intramuscular or intradermal injection although T cell responses were generally lower by intradermal route. These results indicate that DNA vaccines may provide a powerful means to elicit cellular and humoral immune responses against HIV.


Subject(s)
AIDS Vaccines/immunology , DNA, Recombinant , Gene Products, env/immunology , Gene Products, rev/immunology , HIV Antibodies/biosynthesis , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Protein Precursors/immunology , Recombinant Fusion Proteins/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccines, Synthetic/immunology , Animals , Chlorocebus aethiops/immunology , Gene Products, env/genetics , Gene Products, rev/genetics , Genetic Vectors , HIV Antibodies/immunology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp160 , Macaca mulatta/immunology , Mice , Mice, Inbred BALB C , Protein Precursors/genetics , rev Gene Products, Human Immunodeficiency Virus
19.
Nat Med ; 1(6): 583-7, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7585127

ABSTRACT

Vaccination with plasmid DNA expression vectors encoding foreign proteins elicits antibodies and cell-mediated immunity and protects against disease in animal models. We report a comparison of DNA vaccines, using contemporary human strains of virus, and clinically licensed (inactivated virus or subvirion) vaccines in preclinical animal models, to better predict their efficacy in humans. Influenza DNA vaccines elicited antibodies in both non-human primates and ferrets and protected ferrets against challenge with an antigenically distinct epidemic human influenza virus more effectively than the contemporary clinically licensed vaccine. These studies demonstrate that DNA vaccines may be more effective, particularly against different strains of virus, than inactivated virus or subvirion vaccines.


Subject(s)
Antibodies, Viral/biosynthesis , Antigenic Variation/genetics , Antigens, Viral/immunology , DNA, Recombinant/administration & dosage , Hemagglutinins, Viral/immunology , Influenza A virus/immunology , Influenza Vaccines/immunology , Nucleoproteins/immunology , RNA-Binding Proteins , Viral Core Proteins/immunology , Viral Matrix Proteins/immunology , Animals , Antibodies, Viral/immunology , Chlorocebus aethiops , DNA, Recombinant/genetics , Ferrets , Genetic Vectors , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinins, Viral/genetics , Humans , Influenza A virus/genetics , Influenza Vaccines/classification , Influenza, Human/prevention & control , Male , Nucleocapsid Proteins , Nucleoproteins/genetics , Vaccines, Attenuated/immunology , Vaccines, Synthetic/immunology , Viral Core Proteins/genetics , Viral Matrix Proteins/genetics
20.
Curr Opin Biotechnol ; 5(5): 505-10, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7765464

ABSTRACT

Over the past few years, intramuscular injection of non-replicating DNA expression vectors has been demonstrated to be generally applicable as an effective method of producing functional proteins in vivo. This technique has been useful in the study of growth factors, regulation of protein expression, transplantation rejection, gene therapy, immune regulation and the production of monoclonal antibodies. The most successful application of DNA injection has, however, been the generation of immune responses in animal models, with the ultimate goal of developing vaccines for humans. Therefore, this approach has the potential to be a new vaccine technology, in addition to its utility in other areas of research.


Subject(s)
DNA/metabolism , Genetic Vectors , Protein Biosynthesis , Animals , Antigens, CD/biosynthesis , CD4 Antigens/biosynthesis , Cell Line , DNA/administration & dosage , Gene Expression , Gene Products, env/biosynthesis , HIV/genetics , HIV Envelope Protein gp160 , Humans , Mice , Mice, Transgenic , Protein Precursors/biosynthesis , Regulatory Sequences, Nucleic Acid , Repetitive Sequences, Nucleic Acid , Transcription, Genetic , Transfection/methods
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