B cell memory and idiotype network regulation

This investigation examined the clonal dynamics of B-cell expression and evaluated the role of idiotype network interactions in shaping the expressed secondary B-cell repertoire. Three interrelated experimental approaches were applied. The first approach was designed to distinguish between regulatory influences controlled by the major histocompatibility complex (MHC) and regulatory influences controlled by non-MHC factors including the idiotype network. This approach consisted of studies on the clonal dynamics and heterogeneity of the expressed IgG antibody repertoire of BALB/c mice. The second approach involved the analysis of the clonal dynamics of antibody responses of outbred rabbits. This analysis was coupled with studies to detect the occurrence and activity of constituents of the idiotype network. In the third approach the transfer of rabbit lymphocytes from immunized donors to MHC matched naive recipients was used to examine the effects of recipient non-MHC immunoregulatory influences on the expression of donor memory B-cells. Although many memory B cells were unaffected by non-MHC influences, these data show that non-MHC immunoregulatory influences can affect the expression of B-cells in the secondary response of inbred mice and outbred rabbits. The results also indicate that most IgG antibody responses are heterogeneous and are characterized by a stable group of dominant clonotypes. Clonal dominance and B-cell memory were found to be established early in an immune response. The expression of B memory clones appeared to be favored over the expression of virgin B cells. The injection of anti-tetanus antibody induced the antigen independent production of anti-tetanus antibody, probably through idiotypic mechanisms. These results demonstrate that both antibody and antigen can affect the expressed B-ceIl repertoire. Thus, idiotypic interactions are capable of influencing the expression of B-cells and these findings support the existence and function of an idiotype network with strong immunoregulatory potential. ^

Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization.

Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles, useful in medicine and nanotechnology. Their repetitive molecularly-defined architecture is attractive for engineering multivalency, notably for vaccination. However, decorating VLPs with target-antigens by genetic fusion or chemical modification is time-consuming and often leads to capsid misassembly or antigen misfolding, hindering generation of protective immunity. Here we establish a platform for irreversibly decorating VLPs simply by mixing with protein antigen. SpyCatcher is a genetically-encoded protein designed to spontaneously form a covalent bond to its peptide-partner SpyTag. We expressed in E. coli VLPs from the bacteriophage AP205 genetically fused to SpyCatcher. We demonstrated quantitative covalent coupling to SpyCatcher-VLPs after mixing with SpyTag-linked to malaria antigens, including CIDR and Pfs25. In addition, we showed coupling to the VLPs for peptides relevant to cancer from epidermal growth factor receptor and telomerase. Injecting SpyCatcher-VLPs decorated with a malarial antigen efficiently induced antibody responses after only a single immunization. This simple, efficient and modular decoration of nanoparticles should accelerate vaccine development, as well as other applications of nanoparticle devices.

Development of an Interleukin-1β Vaccine in Patients with Type 2 Diabetes

Interleukin-1β (IL-1β) is a key cytokine involved in inflammatory illnesses including rare hereditary diseases and common chronic inflammatory conditions as gout, rheumatoid arthritis, and type 2 diabetes mellitus, suggesting reduction of IL-1β activity as new treatment strategy. The objective of our study was to assess safety, antibody response, and preliminary efficacy of a novel vaccine against IL-1β. The vaccine hIL1bQb consisting of full-length, recombinant IL-1β coupled to virus-like particles was tested in a preclinical and clinical, randomized, placebo-controlled, double-blind study in patients with type 2 diabetes. The preclinical simian study showed prompt induction of IL-1β-specific antibodies upon vaccination, while neutralizing antibodies appeared with delay. In the clinical study with 48 type 2 diabetic patients, neutralizing IL-1β-specific antibody responses were detectable after six injections with doses of 900 µg. The development of neutralizing antibodies was associated with higher number of study drug injections, lower baseline body mass index, improvement of glycemia, and C-reactive protein (CRP). The vaccine hIL1bQb was safe and well-tolerated with no differences regarding adverse events between patients receiving hIL1bQb compared to placebo. This is the first description of a vaccine against IL-1β and represents a new treatment option for IL-1β-dependent diseases such as type 2 diabetes mellitus (ClinicalTrials.gov NCT00924105).Molecular Therapy (2016); doi:10.1038/mt.2015.227.

Microbiota Promotes Chronic Pulmonary Inflammation by Enhancing IL-17A and Autoantibodies

RATIONALE Changes in the pulmonary microbiota are associated with progressive respiratory diseases including chronic obstructive pulmonary disease. Whether there is a causal relationship between these changes and disease progression remains unknown. OBJECTIVE To investigate the link between an altered microbiota and disease, we utilized a model of chronic lung inflammation in specific pathogen free (SPF) mice and mice depleted of microbiota by antibiotic treatment or devoid of a microbiota (axenic). METHODS Mice were challenged with LPS/elastase intranasally over 4 weeks, resulting in a chronically inflamed and damaged lung. The ensuing cellular infiltration, histological damage and decline in lung function were quantified. MEASUREMENTS AND MAIN RESULTS Similar to human disease, the composition of the pulmonary microbiota was altered in disease animals. We found that the microbiota richness and diversity were decreased in LPS/Elastase-treated mice, with an increased representation of the genera Pseudomonas, Lactobacillus and a reduction in Prevotella. Moreover, the microbiota was implicated in disease development as mice depleted of microbiota exhibited an improvement in lung function, reduction in airway inflammation, decrease in lymphoid neogenesis and auto-reactive antibody responses. The absence of microbial cues also markedly decreased the production of IL-17A, whilst intranasal transfer of fluid enriched with the pulmonary microbiota isolated from diseased mice enhanced IL-17A production in the lungs of antibiotic treated or axenic recipients. Finally, in mice harboring a microbiota, neutralizing IL-17A dampened inflammation and restored lung function. CONCLUSIONS Collectively, our data indicate that host-microbial cross-talk promotes inflammation and could underlie the chronicity of inflammatory lung diseases.

Dose-dependent effects of experimental infection with the virulent Neospora caninum Nc-Spain7 isolate in a pregnant mouse model.

Pregnant BALB/c mice have been widely used as an in vivo model to study Neospora caninum infection biology and to provide proof-of-concept for assessments of drugs and vaccines against neosporosis. The fact that this model has been used with different isolates of variable virulence, varying infection routes and differing methods to prepare the parasites for infection, has rendered the comparison of results from different laboratories impossible. In most studies, mice were infected with similar number of parasites (2 × 10(6)) as employed in ruminant models (10(7) for cows and 10(6) for sheep), which seems inappropriate considering the enormous differences in the weight of these species. Thus, for achieving meaningful results in vaccination and drug efficacy experiments, a refinement and standardization of this experimental model is necessary. Thus, 2 × 10(6), 10(5), 10(4), 10(3) and 10(2) tachyzoites of the highly virulent and well-characterised Nc-Spain7 isolate were subcutaneously inoculated into mice at day 7 of pregnancy, and clinical outcome, vertical transmission, parasite burden and antibody responses were compared. Dams from all infected groups presented nervous signs and the percentage of surviving pups at day 30 postpartum was surprisingly low (24%) in mice infected with only 10(2) tachyzoites. Importantly, infection with 10(5) tachyzoites resulted in antibody levels, cerebral parasite burden in dams and 100% mortality rate in pups, which was identical to infection with 2 × 10(6) tachyzoites. Considering these results, it is reasonable to lower the challenge dose to 10(5) tachyzoites in further experiments when assessing drugs or vaccine candidates.

Immunisation with live attenuated Salmonella dublin expressing a sporozoite protein confers partial protection against Theileria parva

Cattle immunised with a recombinant form of p67, the major surface antigen of Theileria parva sporozoites, have been shown to be protected against parasite challenge. In an attempt to simplify the immunisation procedure live attenuated Salmonella strains expressing p67 have been constructed and used to induce anti-p67 immune responses in cattle. All animals immunised with these strains developed strong antibody responses to p67. Specific T cell responses could be detected in the majority of immunised cattle. Challenge with T. parva sporozoites revealed a significant level of protection in immunised calves compared to naive control animals or animals inoculated with non-recombinant attenuated Salmonella.

Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 − T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma

We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte–macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.

An epidemic of tuberculosis with a high rate of tuberculin anergy among a population previously unexposed to tuberculosis, the Yanomami Indians of the Brazilian Amazon

A survey of an emerging tuberculosis epidemic among the Yanomami Indians of the Amazonian rain forest provided a unique opportunity to study the impact of tuberculosis on a population isolated from contact with the tubercle bacillus for millennia until the mid-1960s. Within the Yanomami population, an extraordinary high prevalence of active tuberculosis (6.4% of 625 individuals clinically examined) was observed, indicating a high susceptibility to disease, even among bacille Calmette–Guérin-vaccinated individuals. Observational studies on cell-mediated and humoral immune responses of the Yanomami Indians compared with contemporary residents of the region suggest profound differences in immunological responsiveness to Mycobacterium tuberculosis infection. Among the Yanomami, a very high prevalence of tuberculin skin test anergy was found. Of patients with active tuberculosis, 46% had purified protein derivative of tuberculosis reactions <10 mm; similarly 58% of recent bacillus Calmette–Guérin vaccines exhibited skin test reactions <5 mm. The Yanomami also had higher titers of antibodies against M. tuberculosis glycolipid antigens (>70%) than the control subjects comprised of Brazilians of European descent (14%). The antibodies were mostly of the IgM isotype. Among the tuberculosis patients who also produced IgG antibodies, the titers of IgG4 were significantly higher among the Yanomami than in the control population. Although it was not possible to analyze T-cell responses or patterns of lymphokine production in vitro because of the remoteness of the villages from laboratory facilities, the results suggest that the first encounter of the Yanomami Indian population with tuberculosis engenders a diminished cell-mediated immune response and an increased production antibody responses, relative to other populations with extensive previous contact with the pathogen. These findings suggest that tuberculosis may represent a powerful selective pressure on human evolution that over centuries has shaped the nature of human immune responses to infection.

Phenotypic switching in the human pathogenic fungus Cryptococcus neoformans is associated with changes in virulence and pulmonary inflammatory response in rodents

High-frequency reversible changes in colony morphology were observed in three strains of Cryptococcus neoformans. For one strain (SB4, serotype A), this process produced three colony types: smooth (S), wrinkled (W), and serrated (C). The frequency of switching between colony types varied for the individual colony transitions and was as high as 10−3. Mice infected with colony type W died faster than those infected with other colony types. The rat inflammatory response to infection with colony types S, W, and C was C > S > W and ranged from intense granulomatous inflammation with caseous necrosis for infection with type C to minimal inflammation for infection with type W. Infection with the various colony types was associated with different antibody responses to cryptococcal proteins in rats. Analysis of cellular characteristics revealed differences between the three colony types. High-frequency changes in colony morphology were also observed in two additional strains of C. neoformans. For one strain (24067A, serotype D) the switching occurred between smooth and wrinkled colonies. For the other strain (J32A, serotype A), the switching occurred between mucoid and nonmucoid colonies. The findings indicate that C. neoformans undergoes phenotypic switching and that this process can affect virulence and host inflammatory and immune responses. Phenotypic switching may play a role in the ability of this fungus to escape host defenses and establish chronic infections.

Immunity to retroviral infection: The Friend virus model

Friend virus infection of adult immunocompetent mice is a well established model for studying genetic resistance to infection by an immunosuppressive retrovirus. This paper reviews both the genetics of immune resistance and the types of immune responses required for recovery from infection. Specific major histocompatibility complex (MHC) class I and II alleles are necessary for recovery, as is a non-MHC gene, Rfv-3, which controls virus-specific antibody responses. In concordance with these genetic requirements are immunological requirements for cytotoxic T lymphocyte, T helper, and antibody responses, each of which provides essential nonoverlapping functions. The complexity of responses necessary for recovery from Friend virus infection has implications for both immunotherapies and vaccines. For example, it is shown that successful passive antibody therapy is dependent on MHC type because of the requirement for T cell responses. For vaccines, successful immunization requires priming of both T cell and B cell responses. In vivo depletion experiments demonstrate different requirements for CD8+ T cells depending on the vaccine used. The implications of these studies for human retroviral diseases are discussed.

Potential of lipid core peptide technology as a novel self-adjuvanting vaccine delivery system for multiple different synthetic peptide immunogens

This study demonstrates the effectiveness of a novel self-adjuvanting vaccine delivery system for multiple different synthetic peptide immunogens by use of lipid core peptide (LCP) technology. An LCP formulation incorporating two different protective epitopes of the surface antiphagocytic M protein of group A streptococci (GAS)-the causative agents of rheumatic fever and subsequent rheumatic heart disease-was tested in a murine parenteral immunization and GAS challenge model. Mice were immunized with the LCP-GAS formulation, which contains an M protein amino-terminal type-specific peptide sequence (8830) in combination with a conserved non-host-cross-reactive carboxy-terminal C-region peptide sequence (J8) of the M protein. Our data demonstrated immunogenicity of the LCP-8830-J8 formulation in B10.BR mice when coadministered in complete Freund's adjuvant and in the absence of a conventional adjuvant. In both cases, immunization led to induction of high-titer GAS peptide-specific serum immunoglobulin G antibody responses and induction of highly opsonic antibodies that did not cross-react with human heart tissue proteins. Moreover, mice were completely protected from GAS infection when immunized with LCP-8830-J8 in the presence or absence of a conventional adjuvant. Mice were not protected, however, following immunization with an LCP formulation containing a control peptide from a Schistosoma sp. These data support the potential of LCP technology in the development of novel self-adjuvanting multi-antigen component vaccines and point to the potential application of this system in the development of human vaccines against infectious diseases.

Fatty acids as haptens: exploring the limits of antigenicity

Fatty acids (FAs) are relatively small, hydrophobic and highly mobile molecular structures with vital biological functions and a ubiquitous distribution. Surprisingly, however, they can be rendered immunogenic. We have synthesised a novel immunogen in which dicarboxylic linoleic acid was conjugated to a carrier protein. Dicarboxylic fatty acids (DCA) differ from their normal counterparts only by their possession of a carboxyl group at each end of the molecule. When conjugated to proteins as haptens, they are, therefore, presented to the immune system with a free carboxyl group at the distal end, instead of a methyl group. Polyclonal IgG antibodies raised in response to this unique immunogen could bind not only conjugated hapten with high affinity, but also the equivalent free FA in mono and dicarboxylic form. Similar conjugates constructed from normal FAs produced much weaker antibody responses and could scarcely be considered antigenic at all. The cross-reactivities of the anti-DCA antibodies with FA variants differing in the number, position and configuration of their double bonds showed that the antibody paratope (binding site) was structured to accommodate the hapten in a way that depended on the precise shape of the acyl chain. We suggest that FAs become much more effective as B-cell epitopes when presented with their hydrophilic carboxyl group exposed on the surface of immunogenic conjugates. This type of epitope is determined by the particular double bond pattern of the unsaturated acyl chain, as well as the polar head group. (C) 2003 Elsevier Ltd. All rights reserved.

Toward the development of a synthetic group A streptococcal vaccine of high purity and broad protective coverage

Using native chemical ligation, we synthesized a group A streptococcal. (GAS) vaccine that contained three different GAS M protein peptide epitopes in a chemically well-characterized construct in high purity. Two of the peptide epitopes represented variable amino terminal serotype determinants, and the third represented a carboxyl terminal conserved region determinant of the GAS M protein. We also synthesized a lipid core peptide (LCP) construct containing the same three peptides. Upon immunization of mice, the non-LCP construct only elicited antibody responses to all three epitopes with the use of adjuvant. The LCP construct, however, elicited excellent antibody responses to all three epitopes without the need for any additional adjuvant or carrier. We have synthesized the LCP synthetic vaccine system with good reproducibility.

Group A streptococcal vaccine delivery by immunization with a self-adjuvanting M protein-based lipid core peptide construct

Background & objectives: To develop a broad strain coverage GAS vaccine, several strategies have been investigated which included multi-epitope approaches as well as targeting the M protein conserved C-region. These approaches, however, have relied on the use of adjuvants that are toxic for human application. The development of safe and effective adjuvants for human use is a key issue in the development of effective vaccines. In this study, we investigated the lipid polylysine core peptide (LCP) system as a self-adjuvanting GAS vaccine delivery approach. Methods: An LCP-GAS construct was synthesised incorporating multiple copies of a protective peptide epitope (J8) from the conserved carboxy terminal C-repeat region of the M protein. B10.BR mice were immunized parenterally with the LCP-J8 construct, with or without conventional adjuvant, prior to the assessment of immunogenicity and the induction of serum opsonic antibodies. Results: Our data demonstrated immunogenicity of LCP-J8 when coadministered in complete Freund's adjuvant (CFA), or administered in the absence of conventional adjuvant. In both cases, immunization led to the induction of high-titre J8 peptide-specific serum IgG antibody responses, and the induction of heterologous opsonic antibodies that did not cross-react with human heart tissue proteins. Interpretation & conclusion: These data indicated the potential of a novel self-adjuvanting LCP vaccine delivery system incorporating a synthetic GAS M protein C-region peptide immunogen in the induction of broadly protective immune responses, and pointed to the potential application of this system in human vaccine development against infectious diseases.