Andrew W. Heath

Spontaneous Mutations in the CsrRS Two-Component Regulatory System of Streptococcus pyogenes Result in Enhanced Virulence in a Murine Model of Skin and Soft Tissue Infection

CsrS/CsrR is a 2-component system in Streptococcus pyogenes that negatively regulates hyaluronic capsule and several exotoxins. To detect spontaneous mutations in csrRS, mucoid and large colony variants of M1 strain MGAS166 were isolated from experimental murine skin infections. By use of complementation with a csrRS(+) plasmid, relevant mutations were also detected in 7 of 12 human clinical isolates. The presence of spontaneous mutants in mouse infection was associated with larger, more necrotic lesions. Most spontaneous changes in CsrR resulted from single amino acid substitutions, whereas most csrS mutations were frameshift or nonsense mutations. In 2 instances, IS1548 insertions were found in csrS. Experimental inoculation of mixtures of wild-type (wt) and csrRS(-) bacteria yielded larger, more necrotic lesions than did either strain at twice the inoculum, which suggests that these variants may exhibit pathogenic synergy. Spontaneous emergence of csrRS(-) mutants in vivo enhances the virulence of wt bacteria and increases severity of murine skin infection.

Arrest of B Lymphocyte Terminal Differentiation by CD40 Signaling: Mechanism for Lack of Antibody-Secreting Cells in Germinal Centers

Despite extensive research, the role of CD40 signaling in B cell terminal differentiation remains controversial. Here we show that CD40 engagement arrests B cell differentiation prior to plasma cell formation. This arrest is manifested at a molecular level as a reduction in mRNA levels of secretory immunoglobulin gene products such as mu(s) and J chain as well as the loss of the transcriptional regulator BLIMP-1. Furthermore, the inhibition of B cell differentiation by CD40 engagement could not be overcome by either mitogens or cytokines, but could be reversed by antibodies that interfere with the CD40/gp39 interaction. These data suggest that secretory immunoglobulin is not produced by B cells that are actively engaged by gp39-expressing T cells.

Early myeloid cells are high producers of nitric oxide upon CD40 plus IFN‐γ stimulation through a mechanism dependent on endogenous TNF‐α and IL‐1α

Bone marrow contains nonadherent low‐density wheat germ agglutinin‐positive (Fr3‐WGA+) cells that release large amounts of NO and show natural suppressor activity if stimulated with activated T cells. We have assessed the involvement of CD40‐derived signals in NO production and their cytokine requirements. Production of NO by Fr3‐WGA+ cells in co‐culture with activated T cells is inhibited by a competing CD40 soluble fusion protein. Fr3‐WGA+ cells express the inducible NO synthase (iNOS) and release NO following CD40 plus IFN‐γ activation. Production of NO through CD40 is strictly dependent on endogenous TNF‐α and / or IL‐1α, since it is inhibited by neutralizing these cytokines or blocking the TNF receptor (p55). Both cytokines are transcribed when Fr3‐WGA+ cells are stimulated by CD40 signaling plus IFN‐γ, although TNF‐α remains below detection limits in stimulated Fr3‐WGA+ cell cultures. Phenotypic studies combined with data on intracellular iNOS expression and cell sorting indicate that NO‐producing cells are CD40, CD31 (ER‐MP12), CD11b (Mac‐1)low, ER‐MP20 (Ly‐6C) and Gr‐1 (Ly‐6G) positive, consistent with myeloid progenitors. The results point to early myeloid cells as an important cell source of NO once triggered by activated T cells through CD40 and IFN‐γ‐derived signals, in a mechanism involving the production of TNF‐α and / or IL‐1α.

A potent adjuvant effect of CD40 antibody attached to antigen

There is great potential for novel vaccines based on recombinant proteins and synthetic peptides. Unfortunately these antigens often lack the immunogenicity of whole, killed pathogens used in traditional vaccines. Thus there is strong interest in the identification of immunological adjuvants with low reactogenicity, but high potency, to enhance immune responses and realize the potential of these new vaccine strategies. CD40 antibodies have been shown to have adjuvant effects when administered at very high doses. These large doses are impractical and induce a cascade of cytokine release giving rise to septic shock‐like symptoms, as well as splenomegaly and polyclonal antibody production. We show here that a very small amount of CD40 antibody can exhibit potent adjuvant effects when attached to soluble antigen. The lack of detectable systemic effects indicates that this method may be a powerful and practical means of enhancing the efficacy of recombinant vaccines.

Immunization with an Interferon-γ–gp120 Fusion Protein Induces Enhanced Immune Responses to Human Immunodeficiency Virus gp120

Cytokines, including interferon (IFN)-gamma, can be effective immunologic adjuvants but often lack the potency of other, more reactogenic compounds. On the basis of the observation that attachment of IFN-gamma to antigen could further enhance its adjuvanticity, a chimeric protein involving IFN-gamma and gp120 of human immunodeficiency virus was produced, using varying lengths of amino acid linkers between the two moieties. All resultant fusion proteins appeared to be dimerized, but full IFN-gamma biological activity was present only with the longest, 34-aa linker. Immunization with the fusion protein gave rise to enhanced primary antibody responses to gp120, particularly of the IgG2a subclass. In addition, both T cell proliferation and IFN-gamma production in response to antigen were strongly enhanced by primary immunization with the fusion protein. IFN-gamma fused to antigen is a more potent adjuvant for Th1-like responses than is IFN-gamma mixed with antigen.

Functional activity of CD40 antibodies correlates to the position of binding relative to CD154

In this study we describe the characterization of a panel of 12 anti‐mouse CD40 monoclonal antibodies (mAb). Characterization was performed in terms of antibody‐binding site relative to the CD154 ligand, and the relationship between position and functional outcome of binding. The antibodies divided into three groups. The first were strong inhibitors of CD154 binding, and induced strong proliferative and activation signals to B cells. Two antibodies gave intermediary inhibition and comparable levels of activation. The remaining antibodies were found to bind outside the CD154 binding site and were poor inducers of B‐cell activation. Data presented show a strong correlation between location of mAb binding and the resultant activation signal delivered. This correlation is shown to be independent of the isotype of the antibody involved and of its affinity. Implications of these findings are discussed.

A comparison of oral and parenteral routes for therapeutic vaccination with HSV-2 ISCOMs in mice; cytokine profiles, antibody responses and protection

It is likely that recurrent infections with HSV-2 (or HSV-1) are influenced by local levels of immunity at mucosal surfaces, when virus reactivated from the latent state is infecting mucosal epithelial cells. Increasing the levels of cellular and humoral immunity through immunisation and maintaining such increased levels, may reduce establishment and spread of reactivated virus at the local site, thereby ameliorating recurrent disease symptoms. The use of HSV-2 antigens incorporated into immunostimulating complexes (ISCOMs) for immunisation of mice previously infected with HSV-2 was investigated in the present study. Prophylactic administration of HSV-2 ISCOM vaccine to mice elicits local antibody detectable in nasal washings, serum antibody and the presence of cytokines IL-2, IFN-gamma and IL-4 in supernatants from spleen cell cultures stimulated in vitro with HSV-2 antigens. Use of the same vaccine in mice infected previously with HSV-2, results in increased levels of total and subclass serum ELISA antibody and also increased levels of serum neutralising antibody. Treatment of HSV-2 infected mice with the HSV-2 ISCOM vaccine also induces higher levels of the cytokines IL-2, IFN-gamma and IL-4, in in vitro stimulated spleen cell cultures. Challenge with a lethal dose of HSV-1 showed that mice previously infected with HSV-2 and subsequently given two doses of HSV-2 ISCOMs vaccine were protected.

Use of Herpes Simplex Virus (HSV) Type 1 ISCOMS 703 Vaccine for Prophylactic and Therapeutic Treatment of Primary and Recurrent HSV-2 Infection in Guinea Pigs

The effect of subunit vaccination on the incidence and severity of primary and recurrent genital herpes was investigated in the female guinea pig model of herpes simplex virus (HSV) type 2 genital infection. After prophylactic immunization with zwitterionic detergent-solubilized HSV-1 glycoproteins formulated with alhydrogel or as immunostimulating complex particles, significant reductions in the incidence and severity of primary herpetic illness were observed in both vaccinated groups compared with immunization-naive controls. There was a significant reduction in the incidence of spontaneous herpetic recurrences after administration of HSV-1 antigens formulated as immunostimulatory complexes to guinea pigs in a prophylactic mode (P<.01). Increased levels of both postimmunization and postchallenge ELISA and neutralizing antibodies were significant correlates of protection against primary herpetic disease in a prophylactic scenario. However, no correlation was observed between elevated ELISA or neutralizing antibody levels and protection against recurrent disease following prophylactic or therapeutic administration of HSV-1 subunit vaccines.

Antibody responses, cytokine levels and protection of mice immunised with HSV-2 antigens formulated into NISV or ISCOM delivery systems

The immunogenicity of a type 2 herpes simplex virus (HSV-2) antigen preparation following its formulation into immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) was investigated in a murine model. The immune responses induced by each formulation were characterised by antigen specific total and subclass serum responses, and by lymphocyte proliferation and cytokine (interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-gamma (IFN-gamma)) production by in vitro restimulated spleen cells. The degree of protection afforded to mice by these various HSV-2 vaccine preparations against homologous (HSV-2) and heterologous (HSV-1) challenge infection was also determined. The findings suggest that formulation of the HSV-2 glycoprotein antigens with ISCOM or NISV delivery vehicles, and the methods used to prepare these formulations, influenced the immunogenicity of the final preparation. Higher IgG2a and neutralising antibody levels, IL-2 and IFN-gamma levels and lymphoproliferative responses were noted in mice immunised with the HSV-2 ISCOM formulated vaccine preparation. Furthermore, although HSV-2 antigens formulated in dehydration-rehydration NISV, or entrapped in NISV by freeze-thawing at 30 degrees C (HSV-2 NISV 30), also elicited relatively high antibody, IL-2 and IFN-gamma levels and relatively high lymphoproliferative responses, formulation of HSV-2 antigens by freeze-thawing with NISV at 60 degrees C (HSV-2 NISV 60) did not. There were no differences between any of the HSV-2 vaccine formulations in terms of IL-4 induction in in vitro stimulated spleen cell cultures. Almost complete protection against HSV-2 challenge was afforded by the HSV-2 ISCOM preparation, while partial protection against challenge infection was afforded by the HSV-2 NISV 30 vaccine formulation. The findings are discussed in relation to the nature of the immune mechanisms, particularly Th1- or Th2-like responses, that may be elicited by HSV-2 antigen preparations formulated into various delivery systems and the relevance of these immune responses to protection against HSV infection in the murine model.

Naive T-cell receptor transgenic T cells help memory B cells produce antibody

Injection of the same antigen following primary immunization induces a classic secondary response characterized by a large quantity of high‐affinity antibody of an immunoglobulin G class produced more rapidly than in the initial response – the products of memory B cells are qualitatively distinct from that of the original naive B lymphocytes. Very little is known of the help provided by the CD4 T cells that stimulate memory B cells. Using antigen‐specific T‐cell receptor transgenic CD4 T cells (DO11.10) as a source of help, we found that naive transgenic T cells stimulated memory B cells almost as well (in terms of quantity and speed) as transgenic T cells that had been recently primed. There was a direct correlation between serum antibody levels and the number of naive transgenic T cells transferred. Using T cells from transgenic interleukin‐2‐deficient mice we showed that interleukin‐2 was not required for a secondary response, although it was necessary for a primary response. The results suggested that the signals delivered by CD4 T cells and required by memory B cells for their activation were common to both antigen‐primed and naive CD4 T cells.