Shortening the lipid A acyl chains of Bordetella pertussis enables depletion of lipopolysaccharide endotoxic activity

Whooping cough, or pertussis, is an acute respiratory infectious disease caused by the Gram-negative bacterium Bordetella pertussis. Whole-cell vaccines, which were introduced in the fifties of the previous century and proved to be effective, showed considerable reactogenicity and were replaced by subunit vaccines around the turn of the century. However, there is a considerable increase in the number of cases in industrialized countries. A possible strategy to improve vaccine-induced protection is the development of new, non-toxic, whole-cell pertussis vaccines. The reactogenicity of whole-cell pertussis vaccines is, to a large extent, derived from the lipid A moiety of the lipopolysaccharides (LPS) of the bacteria. Here, we engineered B. pertussis strains with altered lipid A structures by expressing genes for the acyltransferases LpxA, LpxD, and LpxL from other bacteria resulting in altered acyl-chain length at various positions. Whole cells and extracted LPS from the strains with shorter acyl chains showed reduced or no activation of the human Toll-like receptor 4 in HEK-Blue reporter cells, whilst a longer acyl chain increased activation. Pyrogenicity studies in rabbits confirmed the in vitro assays. These findings pave the way for the development of a new generation of whole-cell pertussis vaccines with acceptable side effects

Meningococcal outer membrane vesicles, lipopolysaccharide and innate immunity

Adjuvants are molecules that increase the immunogenicity of an antigen and play a key role in initiating an immune response. Traditional whole cell or outer membrane vesicle (OMV) vaccines already have components that are recognized by pattern recognition receptors (PRRs) on immune cells. On the other hand, increasing knowledge and implementation of new techniques has increased the application of highly purified recombinant proteins and single antigens as vaccines. Although much safer, these single antigen vaccines lack efficient immune stimulants, necessitating separate addition of adjuvants. Lipopolysaccharide (LPS), a dominant component of the Gram-negative bacterial outer membrane, can be a strong activator of the innate immune system and is the basis for novel adjuvants. Neisseria meningitidis, a Gram-negative diplococcus, which has humans as only reservoir of infection, can cause bacterial meningitis and sepsis. Although meningococcal LPS is a potent activator of the PRRs TLR4 and MD-2, in its wild type form it would be too toxic for use in humans. Thus for application as adjuvant, either as internal component of whole cell or OMV vaccines or as addition to purified antigens, modulation of its activity is desirable. We have performed modifications to the basic LPS structure, both by deletion of LPS biosynthesis enzymes and heterologous expression of LPS modification enzymes. This provided us with an array of LPS derivatives with a broad range of TLR4/MD-2 stimulating capabilities and differential cytokine induction. Using these LPS derivatives on cells expressing TLR4/MD-2 from different species we found that changes to the LPS structure can have quite different effects in different species. Thus, extrapolation from animal models when testing LPS molecules must be done with caution. Due to the fact that N. meningitidis is an exclusively human pathogen, many of the host-pathogen molecular interactions are specific for receptors on human cells. For example, the meningococcal opacity (Opa) proteins, which bind to the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of receptors thereby aiding attachment and invasion by the bacterium, do so exclusively to the human versions. Because binding of Opa proteins to CEACAM1 was shown to suppress T cell proliferation in vitro, using animal models may not give an accurate picture of their effects on immunogenicity in Opa-containing OMV vaccines. We have immunized transgenic mice expressing human CEACAM1 with OMVs and found that the Opa-specific immune response is lowered in the presence of human CEACAM1 in vivo. Others found similar effects for the other neisserial proteins factor H binding protein, transferrin binding protein B and Neisseria surface protein A in transgenic mice expressing their cognate receptors, indicating a more general effect of reduced immunogenicity with protein antigens that can bind to their receptor

Expression of human CEACAM1 in transgenic mice limits the Opa-specific immune response against meningococcal outer membrane vesicles.

Item does not contain fulltextOuter membrane vesicles (OMVs) have been extensively investigated as meningococcal vaccine candidates. Among their major components are the opacity (Opa) proteins, a family of surface-exposed outer membrane proteins important for bacterial adherence and entry into host cells. Many Opa-dependent interactions are mediated through the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of receptors. Importantly, binding of Opa to CEACAM1 has been reported to suppress human CD4 T cell proliferation in vitro in response to OMV preparations. This raises the question whether OMV vaccines should contain Opa proteins at all. Until now it has been difficult to answer this question, as the proposed immunosuppressive effect was only demonstrated with human cells in vitro, while immunization experiments in mice are not informative because the Opa interaction is specific for human CEACAM1. In the present study we have used Opa+ and Opa- OMVs for immunization experiments in a human CEACAM1 transgenic mouse model. OMVs were prepared from a meningococcal strain H44/76 variant expressing the CEACAM1-binding OpaJ protein, and from an isogenic variant in which all opa genes have been inactivated. Both the CEACAM1 expressing transgenic mice and their congenic littermates lacking it were immunized twice with the OMV preparations, and the sera were analyzed for bactericidal activity and ELISA antibody titres. Total IgG antibodies against the OMVs were similar in both mouse strains. Yet the titres for IgG antibodies specific for purified OpaJ protein were significantly lower in the mice expressing human CEACAM1 than in the nontransgenic mice. No significant differences were found in bactericidal titres among the four groups. Overall, these data indicate that expression of human CEACAM1 confers a reduced Opa-specific antibody response in vivo without affecting the overall immune response against other OMV antigens

Characterisation of the immunomodulatory effects of meningococcal Opa proteins on human peripheral blood mononuclear cells and CD4+ T cells

Opa proteins are major surface-expressed proteins located in the Neisseria meningitidis outer membrane, and are potential meningococcal vaccine candidates. Although Opa proteins elicit high levels of bactericidal antibodies following immunisation in mice, progress towards human clinical trials has been delayed due to previous findings that Opa inhibits T cell proliferation in some in vitro assays. However, results from previous studies are conflicting, with different Opa preparations and culture conditions being used. We investigated the effects of various Opa+ and Opa- antigens from N. meningitidis strain H44/76 in a range of in vitro conditions using peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells, measuring T cell proliferation by CFSE dilution using flow cytometry. Wild type recombinant and liposomal Opa proteins inhibited CD4+ T cell proliferation after stimulation with IL-2, anti-CD3 and anti-CD28, and these effects were reduced by mutation of the CEACAM1-binding region of Opa. These effects were not observed in culture with ex vivo PBMCs. Opa+ and Opa- OMVs did not consistently exert a stimulatory or inhibitory effect across different culture conditions. These data do not support a hypothesis that Opa proteins would be inhibitory to T cells if given as a vaccine component, and T cell immune responses to OMV vaccines are unlikely to be significantly affected by the presence of Opa proteins