Characterization of humoral immune responses induced by immunization with plasmid DNA expressing HIV-1 Nef accessory protein

Mice immunized with plasmid DNA encoding Nef accessory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies which were maintained for at least 16 months. These antibodies produced in response to Nef-expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide for three of the ten sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.     

The role of type-1 and type-2 T-helper immune responses in HIV-1 vaccine protection

The dichotomy of type-1 and type-2 T-helper (Th) immune responses is thought to be an obstacle to develop Human immunodeficiency virus-type- (HIV-1) vaccines capable of inducing effective cellular as well as humoral immune responses. Macaca mulatta were immunized using two different HIV-1sf2 envelope vaccine strategies, based on either immune-stimulating complexes (ISCOM) or chimeric Fowlpox (FP) vaccines. One month following the third immunization all animals were heterologously challenged with simian/human immunodeficiency virus (SHIVsf13). Vaccinated monkeys, which were protected had the highest levels of both type-1 and type-2 HIV-1 specific T-helper cell (Th) responses in addition to the highest homologous and heterogenous virus neutralizing antibodies. To determine how long Th responses persisted and if they correlated with protection, animals were rechallenged after waiting for four months without re-boosting. Macaques which maintained the highest gp120-specific type-1 (IFN-gamma) responses were protected, while there was evidence of viral clearance in two others. These findings demonstrate the importance of both or mixed type-1 and type-2 Th responses in HIV-1 vaccine induced immunity while suggesting a possible role of persistent type-1 responses in maintaining protective immunity over time.     

Macrophage inflammatory protein-1alpha (MIP-1alpha) expression plasmid enhances DNA vaccine-induced immune response against HIV-1

CD8+ cell-secreted CC-chemokines, MIP-1alpha, and MIP-beta have recently been identified as factors which suppress HIV. In this study we co-inoculated MIP-1alpha expression plasmid with a DNA vaccine constructed from HIV-1 pCMV160IIIB and pcREV, and evaluated the effect of the adjuvant on HIV-specific immune responses following intramuscular and intranasal immunization. The levels of both cytotoxic T lymphocyte (CTL) activity and DTH showed that HIV-specific cell-mediated immunity (CMI) was significantly enhanced by co-inoculation of the MIP-1alpha expression plasmid with the DNA vaccine compared with inoculation of the DNA vaccine alone. The HIV-specific serum IgG1/IgG2a ratio was significantly lowered when the plasmid was co-inoculated in both intramuscular and intranasal routes, suggesting a strong elicitation of the T helper (Th) 1-type response. When the MIP-1alpha expression plasmid was inoculated intramuscularly with the DNA vaccine, an infiltration of mononuclear cells was observed at the injection site. After intranasal administration, the level of mucosal secretory IgA antibody was markedly enhanced. These findings demonstrate that MIP-1alpha expression plasmid inoculated together with DNA vaccine acts as a strong adjuvant for eliciting Th1-derived immunity.     

Strengthening the immune response to typhoid vaccine by subcutaneous RNA administration

The authors demonstrated the adjuvant activity of the RNA preparations injected subcutaneously. In the treatment of mice with the vaccine with a stimulant there was increase of antibody production and of their resistance to infection with typhoid causative agent in comparison with the animals which were only immunized. When the adjuvant was used the vaccine dose could be devreased 2- to 8-fold without any reduction of the immunity formed. RNA injection to the immunized animals on the 25th day after the onset of the experiment produced a revaccinating effect.     

Progress in the development of an HIV-1 vaccine

Containment of the acquired immunodeficiency syndrome (AIDS) epidemic will require an effective human immunodeficiency virus type 1 (HIV-1) vaccine. Accumulating evidence suggests that such a vaccine must efficiently elicit an HIV-1-specific cytotoxic T lymphocyte (CTL) response. Nonhuman primate models will continue to provide an important tool for assessing the extent of protective immunity induced by various immunization strategies. Although replication-competent AIDS viruses attenuated for pathogenicity by selective gene deletions have provided protective immunity in nonhuman primate models, the long-term safety of such vaccines in human populations is suspect. Inactivated virus and subunit vaccines have elicited neither CTLs nor antibodies capable of neutralizing a wide array of patient HIV-1 isolates. Considerable effort is now being focused on evaluating live vector-based vaccine and plasmid DNA vaccine approaches for preventing HIV-1 infection both in animal model and human studies. Our growing understanding of the biology of HIV-1 and immune responses to this virus will continue to suggest improved vaccination approaches for exploration.     

Intestinal immune responses to an inactivated oral enterotoxigenic Escherichia coli vaccine and associated immunoglobulin A responses in blood

An inactivated oral enterotoxigenic Escherichia coli (ETEC) vaccine against ETEC diarrhea was given to 25 adult Swedish volunteers. The vaccine consisted of formalin-killed E. coli bacteria expressing the most common colonization factor antigens (CFAs), i.e., CFA/I, -II, and -IV, and recombinantly produced cholera B subunit (CTB). Immunoglobulin A (IgA) antibody responses in intestinal lavage fluid to CTB and CFAs were determined and compared with corresponding responses in stool extracts and serum as well as with IgA antibody-secreting cell (ASC) responses in peripheral blood. Two doses of vaccine induced significant IgA responses to the different CFAs in lavage fluid in 61 to 87% of the vaccinees and in stool in 38 to 81% of them. The most frequent responses were seen against CFA/I. The magnitudes of the antibody responses against CTB and CFA/I in stool correlated significantly (CTB, P < 0.01; CFA/I, P < 0. 05) with those in intestinal lavage. Intestinal lavage responses against CFAs were best reflected by the ASC responses, with the sensitivity of the ASC assay being 80 to 85%, followed by stool (sensitivity of 50 to 88%) and serum antibody (sensitivity of 7 to 65%) analyses. CTB-specific immune responses were seen in >90% of the vaccinees in all assays.     

Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction

Viral infection and vaccination with DNA both induce similar immune responses to encoded antigens that are produced by the host. The availability of antigens in lymphoid organs is important in generating an immune response to viral challenge. Antigen availability may also be important in the response to DNA vaccines, because immune responses are stronger when antigen is secreted from DNA-transfected cells. We directed antigen to lymphoid organs by vaccination with DNA encoding antigen-ligand fusion proteins. The two ligands examined bind to receptors that are present on high endothelial venule cells of lymph nodes or on antigen-presenting cells. Here we show that both the humoral and the cellular immune responses to a model DNA vaccine were enhanced using either antigen-targeting strategy. Moreover, directing antigen to antigen-presenting cells speeded up, and altered the form of, the immune response. Directing antigen to sites of immune-response induction may represent a generic means of tailoring a potent and effective immune response to a DNA vaccine.     

Augmentation of antigen receptor-mediated responses by histamine H1 receptor signaling

Histamine is considered one of the important mediators of immediate hypersensitivity and inflammation, and acts via G protein-coupled receptors. Here, we report that histamine may affect antigen receptor-mediated immune responses of T and B cells via a signal(s) from histamine H1 receptors (H1Rs). Histamine exhibited enhancing effects on the in vitro proliferative responses of anti-CD3epsilon- or anti-IgM-stimulated spleen T and B cells, respectively, at the culture condition that the fetal calf serum was dialyzed before culture and c-kit-positive cells were depleted from the spleen cells. In studies of histamine H1R knockout mice, H1R-deficient T cells had low proliferative responses to anti-CD3epsilon cross-linking or antigen stimulation in vitro. B cells from H1R-deficient mice were also affected, demonstrating low proliferative responses to B cell receptor cross-linking. Antibody production against trinitrophenyl-Ficoll was reduced in H1R-deficient mice. Other aspects of T and B cell function were normal in the H1R knockout mice. H1R-deficient T and B cells showed normal responses upon stimulation with interleukin (IL)-2, IL-4, CD40 ligand, CD40 ligand plus IL-4, and lipopolysaccharide. Collectively, these results imply that the signal generated by histamine through H1R augments antigen receptor-mediated immune responses, suggesting cross-talk between G protein-coupled receptors and antigen receptor-mediated signaling.     

Induction of systemic and mucosal immune responses to human immunodeficiency virus type 1 by a DNA vaccine formulated with QS-21 saponin adjuvant via intramuscular and intranasal routes

Induction of mucosal and cell-mediated immunity is critical for development of an effective vaccine against human immunodeficiency virus (HIV). We compared intramuscular and intranasal immunizations with a DNA vaccine encoding env of HIV-1 and evaluated the QS-21 saponin adjuvant for augmentation of the systemic and mucosal immune responses to HIV-1 in a murine model. Vaccination via the two routes elicited comparable systemic immune responses, and QS-21 consistently enhanced antigen-specific serum immunoglobulin G2a (IgG2a) production, delayed-type hypersensitivity reaction, and cytolytic activity of splenocytes. Intestinal secretory IgA production and cytolytic activity of the mesenteric lymph node cells are preferentially elicited by intranasal immunization, and QS-21 augmented these activities as well. This adjuvant augmented production of interleukin-2 (IL-2) and gamma interferon (IFN-gamma) associated with decrease in IL-4 synthesis by antigen-restimulated splenocytes. The serum immunoglobulin subtype profile showed a dominant IgG2a response and less strong IgG1 and IgE production in a QS-21 dose-dependent manner. As expected, enhancements of humoral and cell-mediated immune responses by QS-21 were abrogated by treatment with anti-IL-2 and anti-IFN-gamma monoclonal antibodies. These results suggest that the intranasal route of DNA immunization is more efficient than the intramuscular route in inducing mucosal immunity mediated by sIgA and mesenteric lymphocytes. Furthermore, QS-21 is able to act as a mucosal adjuvant in DNA vaccination and demonstrates its immunomodulatory property via stimulation of the Th1 subset. This study emphasizes the importance of the route of immunization and the use of an adjuvant for effective DNA vaccination against HIV-1.     

Kinetics of local and systemic immune responses to an oral cholera vaccine given alone or together with acetylcysteine

Disruption of the hydrogen bonding network at the interface of Escherichia coli transaldolase by substitution of R300 to a glutamic acid residue resulted in a monomeric enzyme at basic pH values, with almost no change in the kinetic parameters. The stability of the R300A and R300E mutants towards urea and thermal inactivation is similar to that of the wild-type enzyme. X-ray analysis showed that no structural changes occurred as a consequence of the side chain replacement. This indicates that the quaternary structure is not required for catalytic activity nor does it contribute significantly to the stability of the enzyme. The results are not consistent with a proposed half-of-the-sites reaction mechanism.     

Murine immune responses to Schistosoma haematobium and the vaccine candidate rSh28GST

Longitudinal studies of Schistosoma haematobium infection in CBA mice revealed a progressive down-regulation of cellular immune responses, as measured by mitogenic and antigenic stimulation of in vitro lymphocyte cultures. Antigen-stimulated production of the Th1 cytokine IFN-gamma by splenocytes increased progressively up to 14 weeks post infection, (four weeks after the onset of parasite egg production), before declining swiftly. Levels of the Th2 cytokine IL-4 in the same cultures remained low until 14 weeks, after which they rose rapidly as IFN-gamma declined. High levels of IL-10 coincided with the peak in IFN-gamma production, suggesting a non Th2-restricted role for this cytokine. Both total and antigen-specific immunoglobulin production confirmed parasite egg deposition as being a major stimulus for host humoral responses. The S. haematobium infection failed to elicit detectable T cell responses to the antifecundity vaccine candidate rSh28GST. However, low levels of antibody were detectable in infected mouse serum and strong IgG and IgA production was induced by vaccination with rSh28GST plus adjuvant.     

Optimization of codon usage of plasmid DNA vaccine is required for the effective MHC class I-restricted T cell responses against an intracellular bacterium

In an attempt to study codon usage effects of DNA vaccines on the induction of MHC class I-restricted T cell responses against an intracellular bacterium, Listeria monocytogenes, we designed two plasmid DNA vaccines encoding an H-2Kd-restricted epitope of listeriolysin O (LLO) of L. monocytogenes, LLO 91-99. One DNA vaccine, p91wt, carries the wild-type DNA sequence encoding LLO 91-99, and the other one, p91mam, possesses the altered DNA sequence in which the codon usage was optimized for murine system. Our read-through analyses with LLO 91-99/luciferase fusion genes confirmed that the optimized 91mam DNA sequence showed extremely higher translation efficiency than the wild-type sequence in murine cells. Consistent with this, i.m. injections of p91mam, but not of p91wt, into BALB/c mice were capable of inducing specific CTL- and IFN-gamma-producing CD8+ T cells able to confer partial protection against listerial challenge. Taken together, these observations suggest that optimization of codon should be taken into consideration in the construction of DNA vaccines against nonviral pathogens.     

Induction of immune responses to HIV-1 by canarypox virus (ALVAC) HIV-1 and gp120 SF-2 recombinant vaccines in uninfected volunteers. NIAID AIDS Vaccine Evaluation Group

OBJECTIVE: To determine the ability of live attenuated canarypox virus expressing HIV antigens to induce CD8+ cytotoxic T-cell responses and to prime for neutralizing antibody responses to boosting with purified recombinant gp120 subunit vaccine. DESIGN: A prospective, double-blind, randomized, immunogenicity and safety study was conducted in healthy adults at low risk for acquiring HIV infection and who were seronegative for HIV. METHODS: CD8+ cytotoxic T-cells directed against Env or Gag expressing target cells were measured after live recombinant canarypox-HIV-1 vaccine priming (vaccine given at days 0, 7, 14 and 21). Neutralizing antibodies were measured after subunit boosting (vaccine given at days 28 and 84). RESULTS: CD8+ CTL were induced in 64% of volunteers by the live recombinant canarypox-HIV-1 vaccine. All volunteers who received two doses of subunit vaccine after live recombinant canarypox priming developed neutralizing antibodies directed against laboratory strains of HIV-1 and seven out of eight volunteers tested developed neutralizing antibodies to the primary isolate, BZ167, but to none of eight other primary isolates. Unprimed controls had low or absent neutralizing antibodies after two doses of subunit vaccine. CONCLUSIONS: The live canarypox vector was safe, stimulated cytotoxic T-cells and primed for a vigorous neutralizing antibody response upon boosting with subunit gp120 vaccine. This vaccine combination should be evaluated further for inducing protection against HIV infection.     

Modulation of viral immunoinflammatory responses with cytokine DNA administered by different routes

The efficacy of plasmid DNA encoding cytokine administered by different routes, systemic or surface exposure, was evaluated and compared for their modulating effects on subsequent lesions caused by infection with herpes simplex virus (HSV). Systemic or topical administration of both interleukin-4 (IL-4) and IL-10 DNA but not IL-2 DNA caused a long-lasting suppression of HSV-specific delayed-type hypersensitivity response. IL-4 or IL-10 DNA preadministration also modulated the expression of immunoinflammatory lesions associated with corneal infection of HSV. Suppression of ocular lesions required that the DNA be administered to the nasal mucosa or ocular surfaces and was not evident after intramuscular administration. The modulating effect of IL-10 DNA was most evident after topical ocular administration, whereas the effects of IL-4 DNA given by both routes appeared to be equal. Preexposure of IL-4 DNA, but not IL-10 DNA, resulted in a significant change in Th subset balance following HSV infection. Our results indicate that the modulating effect of IL-4 or IL-10 DNA may proceed by different mechanisms. Furthermore, our results suggest that surface administration of cytokine DNA is a convenient means of modulating immunoinflammatory lesions.     

Genetic control of immune response to recombinant antigens carried by an attenuated Salmonella typhimurium vaccine strain: Nramp1 influences T-helper subset responses and protection against leishmanial challenge

Attenuated strains of Salmonella typhimurium have been widely used as vehicles for delivery and expression of vaccine antigens in murine models of infectious disease. In mice, early bacterial replication following infection with S. typhimurium is controlled by the gene (Nramp1, formerly Ity/Lsh/Bcg) encoding the natural-resistance-associated macrophage protein (Nramp1). Nramp1 regulates macrophage activation and has multiple pleiotropic effects, including regulation of tumor necrosis factor alpha, interleukin 1beta (IL-1beta), and major histocompatibility complex class II molecules, all of which influence antigen processing and presentation. Nramp1 also has a direct effect on antigen processing, possibly by regulating the activity of proteases in the late endosomal compartment. Hence, there are multiple ways (regulation of bacterial load or recombinant antigen dose, class II molecule expression, costimulatory or adjuvant activity, and antigen processing) that Nramp1 might influence responses to recombinant salmonella vaccines. To test the hypothesis that Nramp1 influences responses to vaccination, congenic mouse strains have been used to analyze immune responses to recombinant antigens (tetanus toxoid antigen and leishmanial gp63) carried by live attenuated S. typhimurium aroA aroD mutants. Results show that congenic mice carrying the wild-type (S. typhimurium resistance) Nramp1 allele mount a predominantly T-helper-1 (IL-2 and gamma interferon) response to vaccination and show enhanced resolution of lesions following challenge infection with Leishmania major. In contrast, mice carrying mutant (S. typhimurium susceptibility) Nramp1 mount a T-helper-2 (immunoglobulin E and IL-4) response and show exacerbated lesion growth upon challenge.     

Immunity to jellyfish venoms: suppression of venom-induced immune responses in ultraviolet B-irradiated mice

Lymph node and spleen cells from mice immunized with sea nettle (Chrysaora quinquecirrha) venom exhibited a proliferative response after exposure to the homologous antigen or that of a related jellyfish, Physalia (Portuguese man-o'-war). Native venom was a more effective stimulant than heated, non-lethal venom. Ultraviolet light treatments administered to the skin either before or after venom sensitization suppressed the proliferative response of these internal immune cells.     

Compartmentalization of Th1/Th2 cytokine responses to experimental Yersinia pseudotuberculosis infection in cats

Specific pathogen-free cats were inoculated subcutaneously into the drainage areas of the left auricular and popliteal lymph nodes with living Yersinia pseudotuberculosis. Inflammation was evident at the inoculation sites and the regional lymph nodes were palpably enlarged at 48 h post-infection. Lymph node enlargement was due to marked paracortical lymphoid hyperplasia and variable neutrophil infiltrates. Yersinia was cultured from the regional lymph nodes and/or spleens of three of the six cats, indicating systemic spread of bacteria. Specific T-helper 1 and 2 (Th1, Th2) cell-associated cytokine mRNA levels were compared in regional lymph nodes, peripheral blood mononuclear cells (PBMC) and spleen at 48 h post-inoculation. Relative to unstimulated control tissues, there was a significant increase in TNF-alpha, IFN-gamma, IL-12, and IL-10 mRNAs in spleen with down-regulation of IL-4. Significant up-regulation of TNF-alpha and down-regulation of IL-4 were also observed in PBMC. Paradoxically, 48 h stimulated lymph nodes showed only minimal differences in cytokine mRNA expression when compared to lymph nodes from mock-inoculated control animals or unchallenged contralateral lymph nodes from the same animal. This study demonstrated that cats, like mice, respond to an intracellular pathogen such as Y pseudotuberculosis with a predominantly Th1-type immune response. The cytokine responses in regional lymph nodes and spleen were asynchronous, while cytokine stimulation in cells of the spleen was mirrored by PBMC.     

CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice

Mucosal immunity is difficult to induce with subunit vaccines unless such vaccines are administered with a mucosal adjuvant such as cholera toxin (CT); however, CT is toxic in humans. Synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG) are potent adjuvants for the induction of Th1-like systemic immune responses against parenterally delivered proteins. Here, we show in mice that intranasal delivery of hepatitis B surface Ag, which alone has no effect, elicits good immune responses when given with CpG oligodeoxynucleotides and/or CT. Overall, CpG is superior to CT for the induction of humoral and cell-mediated systemic immunity as well as mucosal immune responses (IgA) at local (lung) and distant (feces) sites. Furthermore, CpG and CT act synergistically, giving stronger responses than those observed with 10 times more of either adjuvant alone. Ab isotypes were predominantly IgG1 (Th2-like) with CT, mixed IgG1/IgG2a (Th0) with CpG, and predominantly IgG2a (Th1-like) with CpG and CT together.     

Size effect on systemic and mucosal immune responses induced by oral administration of biodegradable microspheres

Induction of systemic and mucosal immune responses following oral administration of biodegradable poly(D,L-lactic acid) (PDLLA) microspheres containing a model antigen, ovalbunin (OVA) was studied using microspheres with different average diameters of 0.6, 1.0, 4.0, 7.0, 11.0, 15.0, 21.0, and 26.0 microns. They were prepared from double emulsion with the solvent evaporation method, followed by size fractionation on counterflow elutriation. OVA was released from the microspheres in vitro over 80 days, irrespective of their size. Production of the serum anti-OVA IgG antibody and secretory OVA-specific IgA antibody in the mice gut was assessed following the oral administration of PDLLA microspheres containing OVA. Microspheres with a diameter of 4.0 microns enhanced the serum antibody in contrast with that of free OVA, but were not effective in inducing the gut secretion of IgA antibody. On the other hand, OVA-containing microspheres with a diameter of 7.0 microns enhanced IgA secretion to a significant extent compared with free OVA, whereas those with 26.0 microns in diameter were ineffective. Body distribution study revealed that the amount of microspheres taken up into Peyer's patches (PP) increased with the increasing size up to 11.0 microns, thereafter decreased, and finally became zero when their diameters were 21.0 microns or larger. The microspheres taken up into PP were translocated to the spleen, but no microspheres were noticed in the spleen when the size was larger than 5 microns. After being taken up inot PP, microspheres < 5 microns in diameter seemed to be transported to the spleen, a systemic lymphoid tissue, where the released antigen stimulated a serum antibody response, but larger microspheres probably remained at PP without being translocated to the spleen over the course of their antigen release, leading to induction of IgA secretion. It was concluded that the body distribution pattern of microspheres following the PP uptake was a key factor to regulate the induction of systemic and mucosal immune responses.     

Psychosocial influences on immune responses to HSV-1 infection in BALB/c mice

The effects of differential housing (one or four mice/cage) on T-helper (Th) cell markers of cellular and humoral immune responses were examined. Differentially housed male BALB/cJ mice were infected with herpes simplex virus (HSV)-1 (Patton strain), and in vitro cytokine production [interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-gamma] by splenocytes and popliteal lymph node cells and serum antibody titers (IgM and IgG) were evaluated. Differential housing of male BALB/c mice influenced the magnitude, but not the kinetics, of some, but not all, immune responses to HSV-1. Splenocytes from individually housed mice produced more IL-2, IFN-gamma, IL-4, and IL-10 than splenocytes from group-housed mice; in popliteal lymph node cells, only IFN-gamma and IL-10 production was influenced by housing. Although the social environment influenced cytokine production, there were no concomitant changes in circulating IgM or IgG antibody titers. These results do not support the hypothesis that dominant Th cell responses are the primary targets of this psychosocial manipulation, or that a reciprocal relationship exists between Th1 and Th2 cell-derived cytokines.