Induction Of Humoral Immune Responses Research Articles

Gene-based vaccines.

The development of vaccines is one of the best examples of the impact of biomedical advances upon human health. But, despite the successes, such as the elimination of smallpox from the planet, there remain a number of diseases for which we have no vaccines, and which infect and often kill millions of people each year (see Table 1). These diseases include those due to newly emerging pathogens such as HIV, as well as ancient scourges such as tuberculosis, where, although there is a vaccine, a better one is needed. A look at the new technologies being used to develop vaccines will be presented here.

Recognition and regulation of metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway mediating induction of humoral immune responses

Proteolytic activity released within an organism by wounded tissues or invading pathogens can strongly impair the physiological homeostasis when it remains non-regulated. Thus, an efficient mechanism that enables recognition and inactivation of non-regulated proteolytic activity is essential to limit toxic effects. In larvae of the Greater wax moth Galleria mellonella we discovered that injection of bacterial thermolysin at a sublethal concentration mediates both acquired resistance against a subsequently injected lethal concentration of this metalloproteinase and stimulation of humoral immune response accompanied by the synthesis of an inducible metalloproteinase inhibitor (IMPI) which is released within the haemolymph. In search of a putative mechanism mediating recognition and regulation of released microbial metalloproteinases we determined that thermolysin-mediated hydrolysis of G. mellonella haemolymph proteins in vitro yields small (<3 kDa), heat-stable molecules which were discovered to represent potent elicitors of humoral immune responses when injected into untreated larvae. Obtained results allowed to design a model explaining for the first time regulation of released metalloproteinases within the haemolymph of insects. The determined coherence between regulation of released metalloproteinases by IMPI and the simultaneous induction of antimicrobial proteins provides a new insight into the mechanisms leading to expression of genes in course of humoral immune responses.

Humoral immune responses to DNA vaccines expressing secreted, membrane bound and non-secreted forms of the: Taenia ovis 45W antigen

The antibody response to DNA vaccines expressing secreted, membrane bound and non-secreted forms of the same antigen was investigated. The antigen gene selected for these studies was the full length 45W antigen gene from Taenia ovis. This gene encodes a host protective membrane bound antigen with a native secretion signal at the amino terminus and a hydrophobic anchor domain at the carboxyl terminus. Full length and rationally truncated forms of the 45W antigen gene were generated and used to construct DNA vaccines encoding membrane bound, secreted and non-secreted forms of the 45W antigen. The cellular localisation of these antigen forms was confirmed by Western blot studies. BALB/c mice were immunised intramuscularly with plasmid DNA and serum antibody responses measured by enzyme linked immunosorbant assay (ELISA). The cellular localisation of DNA vaccine antigen had a significant effect on the magnitude but not the subclass of antibody responses. Immunisation with DNA expressing secreted 45W generated three-fold higher antibody titres than immunisation with DNA expressing membrane bound 45W, and 18-fold higher antibody titres than DNA expressing non-secreted 45W. All mice generated a predominantly IgG1 antibody response indicative of a TH-2 type immune response. These results indicate that the optimal induction of humoral immune responses to intramuscular genetic immunisation with the 45W antigen, requires the active secretion of antigen. This observation may be of value during the design of DNA vaccines in the future.

Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T Helper Epitopes (PADRE) for antibody responses in vivo

Induction of humoral immune responses against protein antigen requires that two independent signals be delivered to B cells. It is currently assumed that simple monovalent synthetic peptides would not be effective immunogens for antibody responses because they would not be anticipated to effectively generate the necessary signals unless conjugated to a complex carrier system. In this study, the immunogenicity of short linear peptide constructs comprising Plasmodium vivax B cell epitopes (PVB) and non-natural Pan-DR T helper cell epitopes (PADRE) was assessed in mice and compared to other types of antigen constructs. The 33-residue PADRE-PVB linear constructs were highly immunogenic and induced responses comparable to those obtained with the multiple antigen peptides (MAP) constructs, both in terms of absolute titers and quality of antibody responses. The anti-PVB antibody responses were of long duration, composed mostly of IgG and reactive with intact sporozoites. The PADRE-PVB constructs were immunogenic when formulated in adjuvants such as Alum and Montanide ISA 51 underlining the relevance of these findings for vaccine development.

In vitro complement-dependent binding and in vivo kinetics of pneumococcal polysaccharide TI-2 antigens in the rat spleen marginal zone and follicle.

For a better understanding of the spleen-dependent induction of the humoral immune response against thymus-independent type 2 antigens, we have studied the in vitro and in vivo localization of different capsular pneumococcal polysaccharides (PPSs) in the rat spleen. In this study, we found that in vitro binding of PPS types 3, 4, 6B, 9N/V, 14, and 23F was dependent on complement (probably a C3 fragment) and that the localization was predominantly restricted to the marginal-zone B lymphocytes and the follicular dendritic cells. In vivo, we observed with increase of time a shift of localized antigens. Shortly after injection, all PPS types localized in the marginal-zone B lymphocytes, then localized in the outer follicular mantle, and finally were found to be diffuse in the complete follicle and follicle corona. PPS types 3 and 9N/V and later also PPS type 23F localized additionally in red pulp macrophages. In particular, the localization in the marginal zone is important since the low flow in this area in combination with strongly CD21+ B cells, which are activated early, gives a maximum opportunity for the induction of a primary humoral immune response with subsequent differentiation into plasma cells or migration to the germinal center. In addition, the localization of PPSs at follicular dendritic cells should be considered important in the induction of an ongoing immune response not restricted to the spleen.

Mucosal and Systemic Immunization using Cochleate and Liposome Vaccines

AbstractFor more than a decade our laboratories have been combining concepts in biochemistry, virology and immunology, in order to develop a conceptual basis for vaccine design. Our long term goals have been to construct simple and well defined immunogens which would stimulate specific immune responses in vivo. Using this approach, we hypothesized that it should be possible to define the structural and biochemical parameters of an immunogen that are necessary and sufficient to stimulate designated effector arms of the immune system. Through the use of covalently coupled peptide complexes, we have been able to define minimal requirements for the induction of humoral immune responses. Minimal requirements for the induction of CD 8 +, MHC Class I restricted Cytotoxic T Lymphocytes have been defined through the use of fusogenic proteoliposomes and simple peptide-lipid complexes (1,2). Finally, we have described a unique, highly stable, lyophilizable, lipid-based vaccine carrier and delivery formulation called...

AK-5 Tumor-Induced Expression of Interleukin-12: Role of IL-12 in NK-Mediated AK-5 Regression

Spontaneous regression of AK-5, a histiocytic tumor, is mediated by CD3 -, CD8 + NK cells through ADCC. The onset of AK-5 regression is associated with the induction of humoral immune response and the augmentation of effector function. The mechanism of tumor cell death involves both necrosis and apoptosis. Interleukin-12, a 75-kDa heterodimeric cytokine, has multiple effects on T and NK cells. We have investigated the role of IL-12 in the NK cell-mediated AK-5 tumor regression process. Subcutaneous transplantation of AK-5 tumor induced the expression of IL-12 (p35 and p40) message by Day 6-8 in the splenocytes of syngenic rats. Similarly, analysis of serum samples from tumor-bearing animals showed the presence of circulating IL-12 around the same time. Interaction of immune cells with antibody-tagged AK-5 cells in vitro also triggered the expression of IL-12 message and protein by 3 hr. The circulating IL-12 in the sera of tumor-rejecting animals, as well as rIL-12, stimulated NK cell proliferation, expression of CD16 and CD25, and the activation of NK cell function. These observations suggest that the ability of the AK-5 tumor to induce the endogenous production of IL-12 may be responsible for keeping the NK cells constantly in an activated state, thus demonstrating an efficient mechanism for the complete regression of the tumor.

Induction of humoral immune response against Plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library

The mouse monoclonal antibody 2A10 (immunoglobulin G), which recognizes the (NANP)n repeat of Plasmodium falciparum circumsporozoite surface protein, was used to screen a filamentous phage epitope library expressing random amino acid hexamers. The sequences obtained were TNRNPQ, SNRNPQ, NND-NPQ, SNYNPQ, and QNDNPQ (single-letter amino acid designation). These peptides showed 50% homology with the native epitope (PNANPN) and therefore were considered to mimic its structure (mimotopes). Two of these mimotopes (TNRNPQ and NNDNPQ) inhibited the binding of monoclonal antibody 2A10 to the recombinant protein R32LR, which contains the amino acid sequence [(NANP)15NVDP]2. Immunization of mice and rabbits using the peptide (TNRNPQ)4 induced a humoral response that recognized R32LR by an enzyme-linked immunosorbent assay and P. falciparum sporozoites by an immunofluorescence assay. These results suggest that phage epitope libraries can be exploited to screen for mimotopes in the design of subunit vaccines against infectious agents.

Multivalent, but not divalent, antigen receptor cross-linkers synergize with CD40 ligand for induction of Ig synthesis and class switching in normal murine B cells. A redefinition of the TI-2 vs T cell-dependent antigen dichotomy.

A number of previous studies have suggested that cross-linkage of the B cell Ag receptor may be critical for induction of humoral immune responses to T cell-dependent (TD) Ags in vivo. Previous work also indicated a critical role, in these responses, for CD40-mediated signaling mediated by binding of the inducible T cell membrane protein, CD40 ligand (CD40L). Data in this manuscript demonstrate that concentrations of bivalent anti-IgD or anti-IgM Ab as high as 30 micrograms/ml induced little if any enhancement of CD40-dependent Ig secretion by resting murine B cells. In contrast, concentrations as low as 3 pg/ml of multivalent, dextran-conjugated, anti-IgD (alpha delta-dex) or anti-IgM (alpha mu-dex) were strongly synergistic with CD40L for induction of B cell proliferation, viable cell outgrowth, Ig isotype switching, and maturation to Ig secretion. As many as 30% of the B cells became membrane IgG1+ after stimulation with CD40L, anti-Ig-dextran, and IL-4 + IL-5, with a concomitant three- to fivefold increase in numbers of viable cells as compared with control cultures. High Ig secretory responses were obtained in response to the combined actions of CD40L and alpha delta-dex or alpha mu-dex, utilizing concentrations of B cell activator that when acting alone induced only modest Ig secretion. Surprisingly, although we previously demonstrated that alpha delta-dex selectively and strongly suppressed IgE production by T cell-activated B cells, it strikingly augmented IgE expression by CD40L-activated B cells. These data suggest 1) a key role for Ag receptor cross-linkage in CD40-dependent induction of humoral immune responses, 2) that to achieve a membrane Ig-dependent enhancing effect in the presence of activated T cells, TD Ags must be displayed to the B cell as a multivalent array of epitopes, 3) that picomolar concentrations of Ag can mediate this effect, and 4) that at least for induction of IgE responses, B cell stimulation via CD40L or via activated T cells may lead to a qualitatively different pathway of activation.

Lipid matrix-based vaccines for mucosal and systemic immunization.

For more than a decade our laboratories have been combining concepts in biochemistry, virology, and immunology in order to develop a conceptual basis for vaccine design. Our long-term goals have been to construct simple and well-defined immunogens that would stimulate specific immune responses in vivo. Using this approach, we hypothesized that it should be possible to define the structural and biochemical parameters of an immunogen that are necessary and sufficient to stimulate designated effector arms of the immune system. Through the use of covalently coupled peptide complexes, we have been able to define minimal requirements for the induction of humoral immune responses (Mannino et al., 1992). This represents a significant advance in eliciting an immune response to peptides, because it requires only peptides and phospholipids in the absence of additional adjuvants. It is different from the previous use of peptides and liposomes since here the peptides are covalently linked to a hydrophobic anchor and integrated into the phospholipid complex, rather than passively adsorbed or encapsulated. The presentation of peptide as part of a peptide-phospholipid complex (in contrast to encapsulation or nonspecific absorption) may be more similar to the natural presentation of an epitope in the context of an in vivo antigenic challenge. This technology also allows us to incorporate B and Th epitopes in a number of forms--as a single peptide, as two peptides in the same liposome, or as a peptide with viral glycoproteins in the same liposome. These data also demonstrate that Th epitopes do not have to be covalently linked to the B-cell epitope in order to provide help for that epitope. The implications of the data reported here are significant for both basic science and applied technologies. In basic science, the peptide-phospholipid complexes are potentially useful for analyzing the cooperative effects of B- and T-cell epitopes in the in vivo immune response. Since the peptide-phospholipid complexes are totally synthetic and highly immunogenic, they may be constructed in any formulation required to answer questions on the roles of B and T cells in promoting an immune response. Furthermore, since the number of antigenic sites is limited only by the number of peptides included in the peptide-phospholipid complexes, these constructs may be useful in producing antisera or monoclonal antibodies to weakly antigenic regions of a large protein, since the lack of antigenic competition should enhance the immunogenicity of these regions. Clinically, this technology will expand the potential for subunit vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)

Protection against ocular and cutaneous infection with herpes simplex virus type 1 by intragastric immunization with live virus.

Intragastric administration of live herpes simplex virus type 1 (HSV-1) was assessed for the induction of humoral immune responses and for protection against ocular and cutaneous challenge with virus. Mice showed no clinical abnormalities following intragastric inoculation with three different strains of virus (Miyama + GC, SC16, and P2C6, a thymidine kinase-defective mutant). Replication of virus was not detected in the oesophagus, superior cervical ganglia or coeliac ganglia of such animals and latent infection was not detected in these ganglia at later times after inoculation. Induction of a mucosal immune response was indicated by the presence of antibody (mainly IgG or IgA)-secreting cells in Peyer's patches. Intragastric immunization gave protection to some extent against ocular challenge and to a greater extent against cutaneous challenge with HSV-1. Following the latter challenge, particularly after intragastric immunization with strains SC16 and Miyama, the establishment of latency was almost completely prevented.

Recent Studies of the Biology of Trypanosoma vivax

Recent biological investigations of the African trypanosomes have been moving away from their previous preoccupation with the phenomenon of antigenic variation. The feeling has arisen that antigenic variation, as demonstrated by the Trypanozoon and Nannomonas subgenera of trypanosomes, is too extensive, the number of serodemes too large and the coexistence of different species in many areas too complicated, to allow any immunoprophylaxis based on antibodies to variable antigens. This is, of course, not to rule out possible biochemical intervention in the biosynthesis or export of VSG molecules by trypanosomes. However, in the case of T. vivax, more information is required concerning antigenic variation and coat structure in this organism before these avenues of investigation are discarded. Ways of improving the yield of mature metacyclic trypanosomes in vitro must be found, so that the contribution of metacyclic variable antigens to the induction of immunity in T. vivax infection can be elucidated. The number of bloodstream VATs must be determined (perhaps by genetic rather than serological means), as there is evidence both for VAT exhaustion contributing to the self-cure of infected hosts, and for a possible limit to the number of VATs which can be expressed in infections in Africa. In South America nothing is known of the number of serodemes of T. vivax which exist, although such knowledge is obviously required, especially if immunity to bloodstream variants is the more important mechanism of inducing immunity to this trypanosome and true cyclical transmission is rare in, or absent from, that subcontinent. Further, in a fragile organism, with a coat of suspect integrity, the method of VSG packing and the relative exposure of underlying surface molecules seems to hold out even more hope for an immunological intervention based on cell surface but invariant molecules than is the case with T. brucei or T. congolense, although this is being attempted with the latter species. In T. brucei infections the appearance of the non-dividing stumpy population acts as a stimulus to the induction of humoral immune responses. In ruminants, antibody responses to T. vivax, at least as judged from lysis tests, lag behind the appearance of the different VATs by some days. It would be important to determine, therefore, whether, if late bloodstream forms could be induced more frequently in the ruminant, the speed of anti-VAT responses could be enhanced. Whilst self-cure appears to be relatively common in T. vivax infections, it is unlikely that it results in sterile immunity.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional characteristics of Peyer's patch lymphoid cells. I. Induction of humoral antibody and cell-mediated allograft reactions.

Peyer's patches from normal mice contain antigen-sensitive B and T cells, but lack the accessory adherent cell type(s) required both for the induction of humoral immune responses and for the induction of allograft reactions against cell surface alloantigens. Immune responsiveness can be restored to cultures of Peyer's patch cells by the addition of either APEC or ME. Peyer's patch B cells can be specifically induced by antigen to synthesize humoral antibody. Peyer's patch T cells can cooperate in B-cell induction and can be induced to mediate an allograft reaction against an allogeneic stimulus. Peyer's patch lymphoid aggregates appear to be a storehouse of antigen-sensitive cells sequestered in such a way as to lack an accessory cell type or factor required for induction,