A prospective, randomized, comparative US trial of a combination hepatitis A and B vaccine (Twinrix) with corresponding monovalent vaccines (Havrix and Engerix-B) in adults.

In an open, randomized, multicenter, controlled clinical trial in the US, 773 adults were administered either a combination hepatitis vaccine (Twinrix: 720 EL.U inactivated hepatitis A antigen and 20 mcg recombinant hepatitis B surface antigen per milliliter) on a 0, 1, 6 month schedule or corresponding monovalent vaccines concurrently (Havrix, 1440 EL.U/ml of hepatitis A antigen at 0, 6 months and Engerix-B, 20 mcg of hepatitis B surface antigen at 0, 1, 6 months). Non-inferiority testing for the primary endpoint, severe soreness, and equivalence testing for the secondary endpoints, anti-HAV seroconversion and anti-HBs seroprotection, showed that safety and immunogenicity were comparable in the two groups.

Vaccination of seronegative volunteers with a human immunodeficiency virus type 1 env/rev DNA vaccine induces antigen-specific proliferation and lymphocyte production of beta-chemokines.

There is a pressing need to test novel vaccine concepts in an effort to develop an effective vaccine for human immunodeficiency virus (HIV) type 1. A phase I clinical study was done to test the immunogenicity of an HIV env/rev DNA vaccine, which was administered intramuscularly to HIV-1-seronegative persons. Subjects received 3 doses of vaccine at a single concentration (100 or 300 microgram) at 0, 4, 8, and 24 weeks. In at least 1 of multiple assays, the 6 subjects who received the 300-microgram dose had DNA vaccine-induced antigen-specific lymphocyte proliferative responses and antigen-specific production of both interferon-gamma and beta-chemokine. Furthermore, 4 of 5 subjects in the 300 microgram-dose group responded to both the rev and env components of the vaccine. The responses did not persist within inoculated individuals and scored in different individuals at different times in the trial. This study supports that HIV-1 DNA vaccine antigens can stimulate multiple immune responses in vaccine-naive individuals, and it warrants additional studies designed to enhance DNA vaccine immunogenicity.

Potential for plasmid DNAs as vaccines for the new millennium.

The advent of new technology and the unmet needs of old and new epidemics of infectious diseases have stimulated a new era of vaccinology. One of the most novel approaches employs plasmid DNA engineered to express one or more genes of the pathogen in mammalian cells. Plasmids may also express cytokine or costimulatory molecules to 'direct' the immune response and/or express altered forms of the antigen to direct it to a specific intracellular compartment or a specific extracellular receptor. The quality of immune responses generated by DNA vaccines in animals has previously only been equaled by live attenuated viral vaccines. The immune stimulating activity of DNA vaccines, combined with their versatility, suggests vast potential for these vaccines.

Enhancement of cellular immune response in HIV-1 seropositive individuals: A DNA-based trial.

A DNA-based vaccine containing HIV-1 Env and Rev genes was tested for safety and host immune response in 15 HIV-infected asymptomatic patients with CD4-positive lymphocyte counts >/=500/microl of blood and receiving no antiviral therapy. Successive groups of patients received three doses of vaccine at 30, 100, or 300 microg at 10-week intervals in a dose-escalation trial. Some changes were noted in cytotoxic T-lymphocyte activity against gp160-bearing targets. Importantly, enhanced specific lymphocyte proliferative activity against HIV-1 envelope was observed in multiple patients. Three of three patients in the 300-microg dose group also developed increased MIP-1alpha levels which were detectable in their serum. Interestingly patients in the lowest dose group showed no overall changes in the immune parameters measured. The majority of patients who exhibited increases in any immune parameters were contained within the 300 microg, which was the highest dose group. These studies support further investigation of this technology for the production of antigen-specific immune responses in humans.

DNA vaccination with HIV-1 expressing constructs elicits immune responses in humans.

Humoral and cellular immune responses have been produced by intramuscular vaccination with DNA plasmids expressing HIV-1 genes, suggesting possible immunotherapeutic and prophylactic value for these constructs. Vaccination with these constructs has decreased HIV-1 viral load in HIV-1-infected chimpanzees. In addition, naive (i.e. non-HIV-1-infected) chimpanzees were protected against a heterologous challenge with HIV-1. Ongoing phase I clinical trials show that therapeutic vaccinations indeed boost anti-HIV-1 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.

First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response.

A DNA-based vaccine containing human immunodeficiency virus type 1 (HIV-1) env and rev genes was tested for safety and host immune response in 15 asymptomatic HIV-infected patients who were not using antiviral drugs and who had CD4+ lymphocyte counts of > or = 500 per microliter of blood. Successive groups received three doses of vaccine (30, 100, or 300 microg) at 10-week intervals in a dose-escalation trial. Vaccine administration induced no local or systemic reactions, and no laboratory abnormalities were detected. Specifically, no patient developed anti-DNA antibody or muscle enzyme elevations. No consistent change occurred in CD4 or CD8 lymphocyte counts or in plasma HIV concentration. Antibody against gp120 increased in individual patients in the 100- and 300-/microg groups. Some increases were noted in cytotoxic T lymphocyte activity against gp160-bearing targets and in lymphocyte proliferative activity. The safety and potential immunogenicity of an HIV-directed DNA-based vaccine was demonstrated, a finding that should encourage further studies.

[3H]1,3-di(2-tolyl) guanidine binds to a sigma 2 receptor on Jurkat cell membranes, but sigma compounds fail to influence immunomodulatory events in human peripheral blood lymphocytes.

The binding characteristics of the sigma ligand [3H]1.3-di(2-tolyl)guanidine (DTG) were investigated in membranes prepared from the Jurkat T cell line. Binding was saturable with a KD of 56 +/- 3 nM and a Bmax of 11706 +/- 3173 fmol/mg protein (n = 3). The rank order of potency for sigma reference compounds to inhibit binding in the Jurkat cell line was ifenprodil > 1,3-di(2-tolyl)guanidine > haloperidol > carbetapentane > (+)3-(3-hydroxyphenyl)-N-propylpiperidine ((+)3-PPP) > (-)pentazocine > caramiphen > (+)pentazocine, and significantly correlated with potency at sigma 2 binding sites in guinea pig brain (r = 0.90, p < 0.01). The immunomodulatory activities of the sigma ligands 1,3-di(2-tolyl)guanidine, haloperidol. (-)pentazocine and (+)pentazocine on CD3-induced proliferation, IL-2 production and Ca2+ flux in human lymphocytes did not reveal any consistent pharmacology that could be ascribed to potency of these compounds at sigma binding sites. Collectively the data demonstrate that the [3H]1,3-di(2-tolyl)guanidine binding site on Jurkat cell membranes has a pharmacology consistent with sigma receptors, but no modulation of functional activity or intracellular events in human peripheral blood lymphocytes correlating with sigma receptors was found.

Novel inhibitors of the nuclear factor of activated T cells (NFAT)-mediated transcription of beta-galactosidase: potential immunosuppressive and antiinflammatory agents.

The preparation of a series of quinazoline-2,4-diones, 1-3, and pyrrolo[3,4-d]pyrimidine-2,4-diones, 4-8 is described. A small number of quinazolinedione analogs were identified from random screening to possess low micromolar (1.3-4.4 microM) potency in the nuclear factor of activated T cells-1-regulated beta-galactosidase expression assay. An expanded analog search resulted in identifying pyrrolopyrimidinedione 4b which is 5-10-fold (0.26 microM) more potent than the quinazolinediones. Replacement of the benzyl group with naphthyl led to greater potency and conformationally restricted analogs 4u-w. The naphthyl and acenaphthyl analogs are 10-100 times more potent inhibitors of beta-galactosidase expression than 4b. Binding affinity data for displacement of radiolabeled 4s from Jurkat cell membranes reflected an excellent correlation with the IC50 value for inhibition of beta-galactosidase activity. These products, whose structure-activity relationships are discussed, are of interest as potential agents for preventing interleukin-2 gene transcription.

Functional characterization of novel IL-2 transcriptional inhibitors.

IL-2-mediated T cell proliferation is a critical early event in the inflammatory process. Formation of the NFAT-1 transcriptional complex on the IL-2 promoter is essential for IL-2 transcription. Using a cell line that is stably transfected with a trimer of the NFAT-1 regulatory element linked to a lac-Z reporter gene, we screened for inhibitors of NFAT-1-mediated beta-galactosidase activity. WIN 61058 and WIN 53071 were identified as microM inhibitors. These compounds also inhibited beta-galactosidase mRNA levels. Similar inhibition of NFAT-1-mediated gene expression was observed in a second cell line, which is stably transfected with NFAT-1 regulatory elements linked to the reporter gene for sCD8. At 10 microM, both compounds inhibited IL-2 mRNA and protein levels in the NFAT-1-linked lac-Z transfectants, and in human lymphocytes. Both compounds inhibited the mixed lymphocyte reaction, and this inhibition was reversed by exogenous IL-2. WIN 53071 inhibited IL-2 production induced in the calcium-dependent PMA and ionomycin pathway. Conversely, calcium-independent anti-CD28 Ab and PMA-induced IL-2 production was resistant. Both compounds altered the NFAT-1 transcriptional complex, causing its retarded mobility on gels. By these functional criteria, we believe we have identified two structurally distinct, novel inhibitors of NFAT-1-mediated transcription.

Expression of human interleukin-2 receptor cDNA in E. coli.

cDNAs for human interleukin-2 receptor were recently cloned and sequenced (Leonard et al., 1984, Nature 311, 626-631; Nikaido et al., 1984, Nature 311, 631-635; Cosman et al., Nature 312, 768-771). In the studies reported here, we describe the expression of a cDNA clone for the human interleukin-2 receptor in E. coli using an "open reading frame" expression vector pMR100. The inserted cDNA was expressed in E. coli transformants as a tripartite fusion polypeptide fused to the lambda cI protein at its amino terminus and to beta-galactosidase at its carboxy terminus. We demonstrate that the bacterially produced IL-2 receptor protein can bind to IL-2.

Adoptive transfer of resistance to growth of an idiotype-secreting hybridoma by T cells from idiotypically suppressed mice.

A/J or CAF1 mice that are suppressed with respect to an idiotype, CRIA, associated with anti-Ar antibodies, and hyperimmunized develop high concentrations of idiotype-suppressor T cells. In this paper we show that such CAF1 mice are resistant to the growth of a CRIA-positive hybridoma that is lethal in normal or in immunized non-suppressed mice. No resistance was observed to the growth of a hybridoma secreting anti-Ar antibodies that lack CRIA. The state of resistance could be adoptively transferred to naive syngeneic recipients with spleen cells or T-enriched spleen cells from suppressed hyper-immunized mice; B-enriched cells were ineffective.

Cell surface phenotype of lymphoid cells from normal mice and mice treated with monoclonal anti-IgD from birth.

Mice treated from birth with mouse monoclonal anti-IgD antibodies develop low frequencies of B cells in the spleen, a small percentage of which express very low levels of sIgD on their cell surface and extremely low frequencies of B cells in their lymph nodes, lacking sIgD entirely. However, the splenic B cells are phenotypically mature in that a high percentage of these cells express Lyb-5, indicating that the expression of sIgD is not a prerequisite for the acquisition of a mature surface antigen repertoire of B cells. In contrast, a high density of sIgM on splenic B cells is expressed, which suggests a predominance of cells with the phenotype of immature B cells and/or activated B cells. Furthermore, the spleen cells from anti-IgD-treated mice lack cells that respond to in vitro stimulation by LPS with an increase in the density of their sIa.

Physiology of Igd. II. Lack of humoral immune responsiveness in lymph nodes of mice treated with anti-IgD from birth.

Previous studies have shown that anti-IgD-suppressed mice give normal primary and secondary splenic plaque-forming cell responses following i.v. challenge, although mice suppressed by the injection of anti-IgD from birth lack IgD-bearing cells in all lymphoid tissue examined. The present studies show that, in contrast, secondary immune responses in regional lymph nodes of such mice, even after i.v. priming with trinitrophenylated B. abortus, respond to a challenge injection in the footpad up to only less than 10% of control levels. When compared with respect to B cell numbers transferred, primed spleen cells from control and anti-IgD-suppressed mice are about equally effective in producing adoptive secondary plaque-forming cell responses in the spleens of recipient mice. Lymph nodes in recipients of anti-IgD-suppressed primed spleen cells show much lower responses than do lymph nodes in recipients of control primed cells, both upon immediate and delayed challenge with antigen in the footpads. It is concluded that the immunodeficiency caused by suppression with anti-IgD is much more marked in peripheral lymph nodes than in the spleen. The possible relationship of these results to the migratory properties of IgD+ as compared to IgD-B cells is discussed.

Induction and persistence of local B cell memory in mice.

Mice were injected with TNP-KLH in one front footpad and challenged in both front footpads 1 to 16 wk thereafter. The response of the contralateral node was significantly lower than that of the ipsilateral node early and again late, but not at intermediate, intervals after priming. This difference was not abolished by boosting with a higher dose of antigen or by administering excess carrier-primed helper T cells before challenge with antigen. The ipsilateral brachial lymph node cells transferred significantly higher B cell memory responsiveness at all intervals after priming than the contralateral lymph node, although at 2 to 4 wk both lymph nodes transferred substantial memory. At later intervals (more than 8 wk) only the ipsilateral lymph nodes transferred significant memory for TNP. Priming in the front footpads did not result in a splenic primary response, but a secondary response was observed in the spleen after boosting in the footpads. The relative importance of antigen localization and circulating memory cells in determining induction and persistence of memory in sites of lymphoid tissue proximal and distal to antigen injection is discussed.

Transfer of memory cells into antigen-pretreated hosts. II. Influence of localized antigen on the migration of specific memory B cells.

Following i.v. injection, 2,4,6-trinitrophenol (TNP)-primed memory cells localized in recipient lymph nodes draining a footpad injection of TNP-hemocyanin (TNP-KLH) in greater numbers than in contralateral nodes draining a p-azobenzenearsonate-coupled KLH injection. Such hapten-specific, unilateral memory B cell localization was still observed in immunosuppressed mice when antigen injections were given as long as 4 days prior to the memory cell transfer. The memory cells could be challenged to form plaque-forming cells by footpad injections of TNP-labeled Brucella abortus at 5 days, but not one day, after cell transfer. The present studies further clarify some parameters of this adoptive memory, as a model for the study of persistent local memory. Measures that promoted the unilateral lymph node retention of 125I-labeled antigen also facilitated unilateral accumulation of TNP-specific memory cells. Such measures included pretreatment of the recipients with cyclophosphamide, rather than gamma irradiation, injection of anti-carrier antibody the day before antigen, or use of small doses of preformed immune complexes instead of antigen alone. In general, a high ratio of lymph node-to-spleen and lymph node-to-blood concentration of antigen in recipients appeared crucial for unilateral localization of memory B cells. Splenectomy of recipients prior to cell transfer enhanced the difference in plaque-forming cell responses between draining and contralateral nodes, but decreased their difference when performed 1 day after cell transfer, suggesting that the spleen may have served as a trap for memory cells. I.v. injection of antigen at the time of B cell transfer also interfered with unilateral localization. The results demonstrate that in the presence of persisting depots of antigen within lymph nodes (and absence of significant amounts of antigen elsewhere), memory B cells can be retained locally without activation into antibody-secreting cells. This mechanism may, therefore, by responsible for the phenomenon of local, humoral, immunological memory.