Inhibition of von Willebrand factor-platelet glycoprotein Ib interaction prevents and reverses symptoms of acute acquired thrombotic thrombocytopenic purpura in baboons.

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) can be explained by the absence of active ADAMTS13, leading to ultra-large von Willebrand factor (UL-VWF) multimers spontaneously interacting with platelets. Preventing the formation of UL-VWF-platelet aggregates therefore is an attractive new treatment strategy. Here, we demonstrate that simultaneous administration of the inhibitory anti-VWF monoclonal antibody GBR600 and the inhibitory anti-ADAMTS13 antibody 3H9 to baboons (prevention group) precluded TTP onset as severe thrombocytopenia and hemolytic anemia were absent in these animals. In addition, partial VWF inhibition was not enough to prevent thrombocytopenia, demonstrating the specificity of this therapeutic strategy. GBR600 treatment of baboons during acute TTP (treatment group) resulted in a rapid recovery of severe thrombocytopenia similar to the platelet count increases observed in TTP patients treated by plasma exchange. Baboons in the control group only injected with 3H9 developed early stages of TTP as previously described. Hence, inhibiting VWF-GPIb interactions is an effective way to prevent and treat the early symptoms of acquired TTP in baboons.

Intranasal immunisation with inactivated RSV and bacterial adjuvants induces mucosal protection and abrogates eosinophilia upon challenge.

We have previously shown that following intranasal exposure to influenza virus, specific plasma cells are generated in the nasal-associated lymphoid tissue (NALT) and maintained for the life of the animal. However, we also showed that following infection with respiratory syncytial virus (RSV), specific plasma cells are generated in the NALT but wane quickly and are not maintained even after challenge, even though RSV-specific serum antibody responses remain robust. Only infection with influenza virus generated sterilising immunity, implying a role for these long-lived plasma cells in protection. We show here that the RSV-specific IgA NALT plasma cell population and lung antibody levels can be substantially boosted, both at acute and memory time points, by intranasal immunisation with inactivated RSV (iRSV) in combination with bacterial outer membrane vesicles (OMV) compared to live RSV alone. Finally, challenge with live RSV showed that immunisation with iRSV and OMV protect against both virus replication in the lung and the eosinophil infiltrate generated by either live RSV or iRSV alone. These data show that immunisation with iRSV and OMV maintains a NALT RSV-specific plasma cell population and generates an efficient protective immune response following RSV infection.

Influenza virus NS1 protein protects against lymphohematopoietic pathogenesis in an in vivo mouse model.

Destruction of peripheral lymphocytes and detrimental alterations in hematopoietic precursors are associated with influenza virus infection in birds and humans. A prominent feature among H5N1 influenza-virus-infected patients with a severe or fatal outcome was found to be lymphopenia and reactive hemophagocytosis. We show here that NS1 protein from human H5N1 influenza isolate A/HK/156/97 reduces both systemic and pulmonary pro-inflammatory cytokines in an in vivo mouse model and protects against bone marrow lymphocyte depletion, an effect which has been shown to be mediated by TNFalpha. These data suggest that the outcome of disease-associated lymphohematopoietic pathogenesis with a pathogenic influenza A virus may depend on the balance between the virus-replication-induced generation of pro-inflammatory cytokines which are a crucial component of the host's anti-viral defense and the ability of the NS1 protein, with or without the interaction of other virus proteins, to counteract such cytokine-mediated adverse effects.

Bone marrow immunosuppression in Salmonella-infected mice is prolonged following influenza virus infection.

OBJECTIVE: It has been shown previously that infection with diverse viruses induces alterations in bone marrow lineage-specific progenitor cells. As complications arising from secondary bacterial infections can adversely affect the host, we investigated whether virally induced hematological alterations could contribute to the enhanced illness observed in such cases. MATERIALS AND METHODS: Mice were infected with influenza virus alone or influenza virus followed by a vaccine strain of Salmonella typhimurium. The effects on hematopoiesis were analyzed by fluorescein-activated cell sorting analysis and immunohistology. RESULTS: Systemic Salmonella typhimurium infection induces depletion of bone marrow erythroid and lymphoid cells. The depletion lasted longer in mice that had been previously infected with influenza virus, compared with mice that had been previously treated with allantoic fluid. Although an increase in splenic lymphoid cells was apparent in the spleens of Salmonella-infected mice, the majority of cells in the enlarged spleens were found to be both immature and mature erythrocytes. CONCLUSION: These results show that bone marrow progenitor cell depletion induced by bacterial infection is prolonged following a viral infection. It is possible that hematological alterations may contribute to the enhanced clinical illness observed in consecutive viral:bacterial infections.

Virus infection-associated bone marrow B cell depletion and impairment of humoral immunity to heterologous infection mediated by TNF-alpha/LTalpha.

We previously showed that influenza virus infection of mice induces a depletion of bone marrow B lineage cells due to apoptosis of early B cells mediated by a mechanism involving TNF-alpha/LTalpha. Here we demonstrate that this effect is also observed with acute lymphocytic choriomeningitis virus (LCMV) infection and resulted in a deficiency of both splenic transitional B cells and mature follicular B cells. To determine whether there was an associated impairment of humoral immunity, we infected mice with LCMV and 10 days later at the peak of the B cell depletion, inoculated them with influenza virus. We found that influenza virus-specific antibody titers were dramatically reduced in mice recovering from LCMV infection compared to those in mice infected with influenza virus alone. Further, we showed that there was no reduction of the influenza virus-specific antibody response in LCMV-infected TNF-alpha/LTalpha-deficient mice, suggesting that TNF-alpha/LTalpha-mediated effects on bone marrow and/or peripheral lymphocytes were responsible for the observed impairment in humoral immunity. These results show that the TNF-alpha/LTalpha production induced following infection with diverse viruses has detrimental effects on early B cells in the bone marrow, and may be among the factors that lead to the severely compromised humoral immunity observed to subsequent heterologous infections.

Altered CD45 isoform expression affects lymphocyte function in CD45 Tg mice.

Transgenic mice have been constructed expressing high (CD45RABC) and low (CD45R0) molecular weight CD45 isoforms on a CD45-/- background. Phenotypic analysis and in vivo challenge of these mice with influenza and lymphocytic choriomeningitis viruses shows that T cell differentiation and peripheral T cell function are related to the level of CD45 expression but not to which CD45 isoform is expressed. In contrast, B cell differentiation is not restored, irrespective of the level of expression of a single isoform. All CD45 trangenic mice have T cells with an activated phenotype and increased T cell turnover. These effects are more prominent in CD8 than CD4 cells. The transgenic mice share several properties with humans expressing variant CD45 alleles and provide a model to understand immune function in variant individuals.

An assessment of different DNA delivery systems for protection against respiratory syncytial virus infection in the murine model: gene-gun delivery induces IgG in the lung.

Immunization with plasmid DNA (pDNA) has the potential to overcome the difficulties of neonatal vaccination that may be required for protection against infection with respiratory syncytial virus (RSV); however, little is known about optimal delivery modalities. In this pilot study we compared mucosal delivery of pDNA encoding RSV F protein encapsulated in poly(DL-lactide-co-glycolide) with delivery of pDNA by gene-gun for the induction of immunity in mice. Intra-gastric or intra-nasal immunization with various doses of microparticles induced weak low levels of RSV-specific serum antibodies in a proportion of mice; in contrast, gene-gun vaccination led to protective immunity associated with a humoral response. Interestingly, RSV-specific antibody was detected in lung fragment cultures following intradermal vaccination with the gene-gun.

Local and systemic antibody responses in mice immunized intranasally with native and detergent-extracted outer membrane vesicles from Neisseria meningitidis.

The mouse humoral immune response toward native or detergent-extracted outer membrane vesicles (NOMVs and DOMVs, respectively) from Neisseria meningitidis was determined after intranasal immunization. Both preparations elicited high frequencies of NOMV-specific antibody-forming cells (AFCs) locally in the nasal associated lymphoid tissue (NALT) after three or four weekly doses. The diffuse NALT (D-NALT) contained ca. 10-fold more NOMV-specific AFCs than those observed in the mediastinal lymph node, spleen, and bone marrow. AFCs observed in the D-NALT were primarily immunoglobulin A positive (IgA(+)) and were maintained for at least 1 month. In contrast, the organized NALT (O-NALT) contained low numbers of AFCs, and the response was relatively short-lived. In other lymphoid tissues, AFCs producing various IgG subclasses and IgM were present with IgG2b-producing AFCs being dominant or codominant with IgA or IgG2a. In serum and in all of the tissues examined, with the exception of the NALT, NOMVs clearly induced a stronger antibody response and a broader range of antibody isotypes than DOMVs. The development of NOMV-specific AFCs in spleen and bone marrow after intranasal immunization was slow compared to intravenous immunization but, once established, the intranasally elicited responses increased steadily for at least 75 days. NOMV-specific antibodies induced via several routes of immunization had high bactericidal activities in serum. Our results indicated that intranasally administered OMVs induced strong local and systemic antibody responses in mice that were relatively long-lived.

Inability to evoke a long-lasting protective immune response to respiratory syncytial virus infection in mice correlates with ineffective nasal antibody responses.

Long-lasting protective antibody is not normally generated in children following primary respiratory syncytial virus (RSV) infection, frequently leading to reinfection. We used the BALB/c mouse model to examine the role of the nasal-associated lymphoid tissue and the bone marrow in the generation of RSV-specific long-lasting plasma cells, with a view to further understanding the mechanisms responsible for the poorly sustained RSV antibody levels following primary infection. We show here that substantial numbers of RSV-specific plasma cells were generated in the bone marrow following challenge, which were maintained thereafter. In contrast, in the nasal-associated lymphoid tissue, RSV-specific plasma cell numbers waned quickly both after primary infection and after challenge and were not maintained at a higher level after boosting. These data indicate that the inability to generate a robust local mucosal response in the nasal tissues may contribute substantially to the likelihood of subsequent reinfection and that the presence of serum anti-RSV antibody without local protection is not enough to protect against reinfection.

Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon.

CD8+ T cell responses can be generated against antigens that are not expressed directly within antigen-presenting cells (APCs), through a process known as cross-priming. To initiate cross-priming, APCs must both capture extracellular antigen and receive specific activation signals. We have investigated the nature of APC activation signals associated with virus infection that stimulate cross-priming. We show that infection with lymphocytic choriomeningitis virus induces cross-priming by a mechanism dependent on type I interferon (IFN-alpha/beta). Activation of cross-priming by IFN-alpha/beta was independent of CD4+ T cell help or interaction of CD40 and CD40 ligand, and involved direct stimulation of dendritic cells. These data identify expression of IFN-alpha/beta as a mechanism for the induction of cross-priming during virus infections.

Bone marrow B cell apoptosis during in vivo influenza virus infection requires TNF-alpha and lymphotoxin-alpha.

Suppression of bone marrow myeloid and erythroid progenitor cells occurs after infection with a variety of different viruses. In this study, we characterize the alterations in bone marrow (BM) lymphocytes after influenza virus infection in mice. We found a severe loss of BM B cells, particularly CD43(low/-)B220(+) pre-B and immature B cells, in influenza virus-infected mice. Depletion of BM B lineage cells resulted primarily from cell cycle arrest and most likely apoptosis within the BM environment, rather than from increased trafficking of BM emigrants to peripheral lymphoid tissues. Use of gene-knockout mice indicates that depletion of BM B cells is dependent on TNF-alpha, lymphotoxin-alpha, and both TNF receptors, TNFR1-p55 and TNFR2-p75. Thus, TNF-alpha and lymphotoxin-alpha are required for loss of BM B lineage cells during respiratory infection with influenza virus.

Cross-reactive polyclonal antibodies to the inner core of lipopolysaccharide from Neisseria meningitidis.

Sera from mice immunized with native or detergent-extracted outer membrane vesicles derived from lipopolysaccharide (LPS) mutant 44/76(Mu-4) of Neisseria meningitidis were analyzed for antibodies to LPS. The carbohydrate portion of 44/76(Mu-4) LPS consists of the complete inner core, Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->5KDO[4-->2 alpha KDO]. Immunoblot analysis revealed that some sera contained antibodies to wild-type LPS which has a fully extended carbohydrate chain of immunotype L3,7, as well as to the homologous LPS. Sera reacted only weakly to LPS from 44/76(Mu-3), which lacks the terminal glucose of the inner core. No binding to more truncated LPS was observed. Consequently, the cross-reactive epitopes are expressed mainly by the complete inner core. Dephosphorylation of wild-type LPS abolished antibody binding to LPS in all but one serum. Thus, at least two specificities of cross-reactive antibodies exist: one is dependent on phosphoethanolamine groups in LPS, and one is not. Detection of these cross-reactive antibodies strongly supports the notion that epitopes expressed by meningococcal LPS inner core are also accessible to antibodies when the carbohydrate chain is fully extended. Also, these inner core epitopes are sufficiently immunogenic to induce antibody levels detectable in polyclonal antibody responses. Meningococci can escape being killed by antibodies to LPS that bind only to a specific LPS variant, by altering the carbohydrate chain length. Cross-reactive antibodies may prevent such escape. Therefore, inner core LPS structures may be important antigens in future vaccines against meningococcal disease.