Biomedical subjects

J Hinkula

Publications and source records attributed to J Hinkula.

At least 37 records · Page 2Linked to original sources

PubMed2000

DNA-encoding enzymatically active HIV-1 reverse transcriptase, but not the inactive mutant, confers resistance to experimental HIV-1 challenge.

The present study was undertaken to examine the immunogenicity of a single plasmid DNA representing the reverse transcriptase (RT) of HIV-1. Plasmids containing the enzymatically active RT as well as a mutated nonenzymatically active RT with nucleotide (nt)-binding motifs of YMDD and YMLL, respectively, were used to immunize mice. Both constructs induced similar good antibody and T cell responses, with a tendency towards antibody directed to peptides representing the active and mutated sites. Immunized mice were challenged with a murine pseudotype HIV-1/MuLV infected spleen cells. Seven out of 10 mice immunized with RT had no recoverable HIV-1, while 10 individuals immunized with the RT mutant and all the 18 controls had high levels of recoverable HIV-1. This indicates that mutation of RT reduces the desired immunogenicity.

AIDS Vaccines↗

PubMed1999

A long-term memory obtained by genetic immunization results in full protection from a mammary adenocarcinoma expressing an EBV gene.

We have tested the capability of a plasmid DNA (pDNA) expressing the EBV nuclear Ag-4 (EBNA-4) to evoke a T cell response-associated protective immune response against a tumor expressing this gene. We have found that ACA mice immunized with EBNA-4-expressing plasmid were partially protected against syngeneic mammary carcinoma line (S6C) expressing EBNA-4 (S6C-E4). This protection was enhanced by coimmunizing mice with EBNA-4- and GM-CSF-expressing plasmids, and a full protection was achieved by coimmunizing mice with EBNA-4- and IFN-gamma-expressing plasmids. Furthermore, mice that have rejected the EBNA-4-positive tumor were also resistant against a subsequent challenge with the original nontransfected tumor line. We then checked for the ability of pDNA immunization to provide a protective long-term memory response. We indeed found that even after 3 mo from the last immunization, full protection was obtained by this method, as compared with full tumor outgrowth in the control-immunized group. These findings support the concept that a nonviral, pDNA-based vaccination strategy is useful to fully protect from the outgrowth of tumors expressing this EBV gene.

Adenocarcinoma↗

PubMed1999

Induction of cytotoxic T-cell responses by gene gun DNA vaccination with minigenes encoding influenza A virus HA and NP CTL-epitopes.

Cytotoxic T-lymphocyte (CTL) response is an important component of anti-viral immunity. CTLs are specific to short peptides presented by MHC-I molecules and immunisation with the exact peptide sequence introduced in the cytosol is therefore a minimal approach, which potentially affords a high degree of controllability. We have examined the induction of murine CTL's by this approach using DNA plasmid minigene vaccines encoding known mouse K(k) minimal CTL epitopes (8 amino acids) from the influenza A virus hemagglutinin and nucleoprotein. We here report that such an approach is feasible and that wild type influenza virus flanking amino acid sequences can influence the CTL response but are not essential for optimal CTL induction. We also examined the effect of different new amino acid sequences flanking the CTL epitopes. In one version, two CTL epitopes were linked together as 'string of beads'. This did not improve CTL induction. In another version, one CTL epitope was inserted into a known T-helper protein (HBsAg). This did significantly augment the response probably due to immunological help from HBsAg Th epitopes. Finally, the CTL inducing minigene DNA vaccines were compared with Flu-induced CTL responses and tested for their protective effect against a lethal influenza A virus infection in mice and no effect was found. We conclude that a specific and highly directed CTL induction is possible by unlinked minigene DNA immunisation, but that CTL induction solely is not always sufficient to provide protection.

Amino Acid Sequence↗

PubMed1999

Dose-response resistance to HIV-1/MuLV pseudotype virus ex vivo in a hairpin ribozyme transgenic mouse model.

We have investigated the efficacy of a hairpin ribozyme targeting the 5' leader sequence of HIV-1 RNA in a transgenic model system. Primary spleen cells derived from transgenic or control mice were infected with HIV-1/MuLV pseudotype virus. A significantly reduced susceptibility to infection in ribozyme-expressing transgenic spleen cells (P = 0.01) was shown. Variation of transgene-expression levels between littermates revealed a dose response between ribozyme expression and viral resistance, with an estimated cut off value below 0.2 copies of hairpin ribozyme per cell. These findings open up possibilities for studies on ribozyme efficacy and anti-HIV-1 gene therapy.

Animals↗

PubMed1999

Immune responses in asymptomatic HIV-1-infected patients after HIV-DNA immunization followed by highly active antiretroviral treatment.

Intensive chemotherapy is capable of reducing the viral load in HIV-1-infected individuals while infected cells are still present. A special property of DNA immunization is to induce both new CTL and Ab responses. We evaluated the possibility of inducing new immune responses in already infected individuals by means of DNA constructs encoding the nef, rev, or tat regulatory HIV-1 genes. Significant changes in viral loads and CD4+ counts were observed in four patients who started highly active antiretroviral treatment (HAART) during the immunization study. The DNA immunization induced Ag-specific T cell proliferation, which persisted up to 9 mo after the last DNA injection, and cytolytic activities but did not, by itself, reduce viral load. Increased levels of CTL precursor cells were induced in all nine DNA-immunized patients. The profile of IFN-gamma secretion observed when human PBMC were transfected with the nef, rev, and tat DNA resembled that found in the CTL activity (nef > tat > rev). Ab responses that occurred after immunizations were of a low magnitude. In accordance with the high IL-6 production induced by the nef DNA plasmid, IgG titers were highest in patients immunized with nef DNA. The initiation of HAART appears to contribute to the induction of new HIV-specific CTL responses, but by itself did not cause obvious re-induction of these activities.

AIDS Vaccines↗

PubMed1999

Induction of mucosal IgA by a novel jet delivery technique for HIV-1 DNA.

Novel ways of delivering plasmid DNA to elicit humoral IgA, IgG and cell-mediated immune responses in mice were investigated. Intraoral administration of DNA in the cheek, using a jet immunization technique, elicited the highest IgA mucosal responses. Intranasal immunization gave strong mucosal IgA responses and persistent systemic IgG. Immunoglobulin isotype analysis revealed an IgG1 profile for intramuscular tongue and gene gun immunizations and an IgG2a profile following oral jet injection and intranasal application. The route of delivery was of importance for the characteristics and quality of the mucosal immune response following DNA immunization. For DNA vaccine delivery, the intraoral jet injection technique has the advantages of being a simple and rapid way of administering the DNA in solution and of provoking specific mucosal IgA when administered in the mucosal associated lymphoid tissue.

Administration, Intranasal↗

PubMed1999

Eukaryotic expression of enzymatically active human immunodeficiency virus type 1 reverse transcriptase.

Reverse transcriptase of human immunodeficiency virus type I is a vitalenzyme in the HIV-1 replication cycle and an attractive target of attempts to arrest a primary viral infection. We designed a vector for eukaryotic expression of the 66 kDa subunit of reverse transcriptase under the control of the immediate early cytomegalovirus promoter. Efficient transient expression of the 66 kDa subunit of reverse transcriptase was achieved in a variety of cells. Immunostaining of the transfected cells revealed the cytoplasmatic localization of reverse transcriptase. Reverse transcriptase activity was detected in all transfected cell lines. Injection of this plasmid encoding the 66 kDa subunit of reverse transcriptase into mice resulted in strong reverse transcriptase-specific immune responses indicating that the 66 kDa subunit of reverse transcriptase is expressed in vivo. Sera from DNA-immunized mice inhibited reverse transcription in vitro.

3T3 Cells↗

PubMed1999

DNA-plasmids of HIV-1 induce systemic and mucosal immune responses.

DNA-based immunization has been shown to induce protective immunity against several microbial pathogens including HIV-1. Several routes of DNA vaccination have been exploited. However, the properties of the immune responses seem to differ with the different routes used for DNA delivery, ultimately affecting the outcome of experimental challenge. We measured the primary immune response following one vaccination. This report presents differences associated with three different DNA delivery routes: intramuscular injection, intranasal application, and gene-gun based immunization. Induction of systemic humoral immune responses was achieved most efficiently by either intranasal or gene-gun mediated immunization, followed by intramuscular injection. Mucosal IgA was reproducibly induced by intranasal instillation of the DNA, and found in lung washings, faeces, and vaginal washings. Cytotoxic T cells were not induced by a single immunization, but were observed after three immunizations using intramuscular injections.

AIDS Vaccines↗

PubMed1999

Long-term immunotherapy in HIV infection, combined with short-term antiretroviral treatment.

Forty asymptomatic HIV-infected individuals with CD4+ lymphocyte levels above 400x10(6)/l were immunized over 5 years with recombinant envelope glycoprotein gp160 (rgp160). After 5 years there was a trend towards more non-progressors in the immunized group as compared to the matched controls. Since immunizations could activate HIV, the first 6 immunizations were followed by 2 weeks of zidovudine or placebo, double-blind. The viral load did not change during the first 6 months and was not different from that of the matched controls after 5 years. The best effect on CD4+ lymphocyte development was seen in individuals with a high viral load randomized to rgp160+zidovudine and in individuals with a low viral load randomized to rgp160+placebo. We conclude that rgp160 is safe and results in temporarily improved CD4+ development. Concomitant antiviral treatment might be of benefit, especially in patients with a more advanced disease and can today be given with more effective combinations.

AIDS Vaccines↗

PubMed1999

Humoral and cell-mediated immune responses in humans to the NSP4 enterotoxin of rotavirus.

Rotavirus nonstructural protein NSP4 has recently been suggested to function as a viral enterotoxin and play a role in the pathophysiological mechanism whereby rotaviruses induce diarrhea. The ability of rotavirus NSP4 to stimulate a humoral immune response was examined in naturally infected children and adults, rotavirus vaccinated children, as well as a cellular immune response in adults. In this study, 10 of 10 naturally infected children and 9 of 10 rotavirus-vaccinated children showed a weak humoral IgG immune response to recombinant NSP4 (rNSP4) and/or a synthetic peptide corresponding to residues 114-134 of NSP4. Modest serum IgG antibody responses were detected in 20 of 20 adults. A cellular immune response to rNSP4 and/or NSP4(114-134) were detected in 8 of 10 adults measured either as a T-cell proliferative response (7 of 10), an increased production of IL-2 (6 of 10), or increased production of interferon-gamma (8 of 10). These results indicate that NSP4 induces a humoral immune response in humans and show for the first time that NSP4 stimulates a cellular immune response, possibly including cytolytic T-cells.

Adult↗

PubMed1998

Immune responses but no protection against SHIV by gene-gun delivery of HIV-1 DNA followed by recombinant subunit protein boosts.

The efficacy of combining immunization with human immunodeficiency vitus type 1 (HIV-1) DNA and HIV-1 recombinant proteins to obtain protection from chimeric simian/human immunodeficiency virus (SHIV) was determined. Four cynomolgus monkeys received four gene-gun immunizations intraepidermally of plasmid DNA encoding HIV-1lai env (gp160), gag, tat, nef, and rev proteins. Ten micrograms of DNA was used per immunization. The animals were boosted twice intramuscularly with 50 microgram of HIV-1lai Env (MicroGeneSys), Gag, Tat, Nef, and Rev recombinant proteins mixed in Ribi adjuvant. The antibody responses were amplified following the administration of the recombinant subunit boosts. One month after the final subunit immunization, the vaccinated animals together with four control animals were challenged intravenously with 10 monkey infectious doses of SHIV that expresses the env, tat and rev genes of HIV-1 and gag and nef from SIV. However, only low titers of neutralizing antibodies were present at the day of challenge. The consecutive HIV-1 DNA and recombinant protein immunizations induced B- and T-cell responses but not protection against SHIV replication nor reduction of the viral load.

AIDS Vaccines↗

PubMed1998

Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients.

BACKGROUND: DNA vaccination is known to generate immune responses against HIV-1 in animal models. We aimed to assess the efficacy of DNA vaccination in induction of immune responses in HIV-1-infected human beings. METHODS: Nine symptom-free HIV-1-infected patients were immunised with DNA constructs encoding the nef, rev, or tat regulatory genes of HIV-1. The patients were selected for having no or low antibody reactivities to these antigens. HIV-1-specific cytotoxic T-lymphocytes (CTLs), precursor frequencies, and antigen-specific proliferative responses were measured before, during, and after three immunisations over 6 months. FINDINGS: Cellular immune reactivities against the HIV-1 regulatory proteins were absent or low before DNA immunisation. DNA vaccination induced detectable memory cells in all patients and specific cytotoxicity in eight patients. CTLs were MHC-class-I restricted and mainly of CD8+ origin. In three patients the cellular activity was transient, decreasing after an initial response. INTERPRETATION: DNA immunisation with HIV-1 genes can induce specific cellular responses in human beings with no apparent side-effects. It is theoretically possible that HIV-1-specific cytotoxic responses to regulatory proteins could lead to infected cells being eliminated before they have released new viral particles. However, it is possible that the patients we selected responded less than would non-selected or non-infected individuals. The small number of patients presented here does not allow generalisation of our findings.

AIDS Vaccines↗

PubMed1998

Seroreactivity to HIV-1 V3 subtypes A to H peptides of Argentinian HIV-positive sera.

Serologic assays could be useful for determining circulating subtypes in different geographic regions. A total of 175 serum samples from the same number of Argentinian HIV-infected patients from Buenos Aires and Rosario were tested against a panel of peptides representing V3 consensus subtypes A through H. A V3 peptide enzyme immunoassay was used for screening the sera. Most sera were reactive with peptides representing subtypes B (58.28%), F (13.14%), and A (8.57%). Cross-reactivity between the remainder of the peptides was observed. Genotypes of eight patients from Rosario were determined and compared with serotyping. Results showed that seven of eight genotyped patients reacted with their respective consensus B peptide and one reacted with consensus B and F. V3 peptide serology proved to be useful for determining HIV-1 clades circulating in Argentina.

Amino Acid Sequence↗

PubMed1998

Induction of specific T-cell responses in HIV infection.

OBJECTIVES: To induce recovery of HIV-1-specific immune responses by combining immunization with antiviral chemotherapy. DESIGN: Forty HIV-infected patients entered a double-blind study with recombinant gp160 in combination with zidovudine or placebo. The pretreatment observation period was around 2 years and the treatment period 5 years. Eighty matched HIV-infected patients served as controls. METHODS: Immune status was monitored by proliferation assays with HIV-specific antigens, mitogens and recall antigens. Viral load, CD4 cell counts, apoptosis, T-cell clonal analysis and CC-chemokine receptor (CCR)-5 status were determined. RESULTS: All immunized patients showed a strong and HIV-specific T-cell proliferative response. This response was related to the immunizations, and was not enhanced by the zidovudine monochemotherapy given during the first 6 months of the immunizations. The treatments did not significantly alter viral load. Potent antiviral combination therapy given to non-immunized individuals reduced their viral load but did not influence HIV-specific immune responses. There was a trend for an increased frequency of non-progression in the immunized group compared with controls. These individuals had both wild-type and mutant CCR-5 genes. CONCLUSION: The results clearly show that restoration of HIV-specific T-cell immunity occurs after immunization with the HIV gp160 antigen and is not influenced by the addition of antiviral monochemotherapy. Even intensive chemotherapy alone did not restore HIV-specific immunity and immunization alone did not influence viral load. This suggests that combinations of intensive chemotherapy with specific HIV immunization would result both in viral load reduction and improved immune responses to HIV.

AIDS Vaccines↗

PubMed1997

Nucleic acid vaccination with HIV regulatory genes: a combination of HIV-1 genes in separate plasmids induces strong immune responses.

The concept of combining several genes in order to immunize against a microbial agent has been tested. We selected human immunodeficiency virus (HIV) genes that individually have been shown to mediate immune responses against HIV proteins. These proteins were the regulating genes/proteins of HIV-1 rev, tat and nef as well as structural genes for gp160 under the control of rev, and the capsid p24 represented by the larger precursor gene p37. Two findings were of particular interest. The combination of these five gene constructs gave strong reactivity to all of them, compared with previous results using each one in single injections. The intranasal immunization route gave good mucosal reactivity by inducing IgG, IgA and T-cell proliferative responses.

AIDS Vaccines↗

PubMed1997

Induction of HIV-1 specific mucosal immune responses by DNA vaccination.

DNA vaccination has been shown to induce immunity against several different pathogens including HIV-1. The authors demonstrate here that administration of DNA vaccines via the intranasal route is sufficient to induce immune responses both at distal mucosal sites and systemically. Since transmission of HIV-1 occurs largely across mucosal surfaces, the intranasal route provides a further means of application for DNA immunization.

Administration, Intranasal↗

PubMed1997

Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes.

Mice immunized with the regulatory genes nef, rev, and tat from human immunodeficiency virus type 1 developed both humoral and cellular immune responses to the gene products Nef, Rev, and Tat. This study demonstrates that it is feasible to induce immune reactions to all of these regulatory gene products. Humoral responses were seen after DNA boosts, while potent T-cell proliferative responses were noted already after a single immunization. A Th1-directed immune response was demonstrated early after immunization. A 3- to 75-fold-stronger T-cell response was seen in animals receiving DNA epidermally compared to that in animals receiving intramuscular injections. Nef, Rev, and Tat putative B- and T-cell epitopes were clearly mapped by using peptides derived from the regulatory proteins and were similar to those which are detected in human immunodeficiency virus infection. Although immunization by the Nef, Rev, and Tat proteins raised high immunoglobulin G titers in serum, the epitope spreading appeared broader after DNA immunization. The combination of all of these regulatory genes together with two genes for structural proteins, the envelope and gag genes, demonstrated that a combined approach is feasible in that reactivities to all antigens persisted or were even augmented. No interference between plasmids was noted.

AIDS Vaccines↗

PubMed1997

Inhibition of early and late events of the HIV-1 replication cycle by cytoplasmic Fab intrabodies against the matrix protein, p17.

BACKGROUND: The HIV-1 matrix (MA) protein, p17, contains two subcellular localization signals that facilitate both nuclear import of the viral preintegration complex early during infection and virus particle assembly late in infection. The dual role of MA in both the afferent and efferent arms of the HIV-1 life cycle makes it an important target for intracellular immunization-based gene therapy strategies. MATERIALS AND METHODS: Here we report, using a new bicistronic vector, that an intracellular Fab antibody, or Fab intrabody, directed against a carboxy-terminal epitope of MA from the Clade B HIV-1 genotype, can inhibit HIV-1 infection when expressed in the cytoplasm of actively dividing CD4+ T cells. RESULTS: Marked inhibition of proviral gene expression occurred when single-round HIV-1 CAT virus was used for infections. In challenge experiments using both laboratory strains and syncytium-inducing primary isolates of HIV-1, a substantial reduction in the infectivity of virions released from the cells was also observed. CONCLUSIONS: This novel strategy of simultaneously blocking early and late events of the HIV-1 life cycle may prove useful in clinical gene therapy approaches for the treatment of HIV-1 infection and AIDS, particularly when combined with genetic or pharmacologic-based strategies that inhibit other HIV-1 target molecules simultaneously.

Animals↗