Comprehensive prevention of meningococcal disease has not been possible due to the lack of a vaccine against organisms associated with the serogroup B capsular polysaccharide. Most global serogroup B disease is caused by just four hyperinvasive genetic groups (the ST-8, ST-11, ST-32 and ST-44 clonal complexes). It was established that these complexes are stably associated with combinations of highly immunogenic Opacity associated (Opa) surface proteins involved in colonisation and invasion of human tissues. 

In a Meningitis UK funded project, optimisation of the expression and purification of several Opa protein variants has taken place. ‘Proof in principle’ has now been achieved that combinations of Opa proteins specific to hyperinvasive genotypes elicit high titres of bactericidal antibody in mice.

To address critical safety and efficacy questions, the current study aimed:

1) to complete extensive characterisation of the murine immune response against the current panel of purified, refolded recombinant Opa proteins as originally funded by Meningitis UK.

2) to systematically investigate the immunomodulatory effects of Opa proteins on proliferation of native human B cells and CD4+ T-cells, on the secretion of pro-inflammatory cytokines from human macrophages and CD4+ T-cells and on the secretion of antibody from B cells.

3) to evaluate the vaccine candidacy of non-native forms of Opa proteins (unfolded recombinant proteins and in vitro manufactured peptides), which could be more advantageous in manufacture and regulation, and to explore their effects on aspects of cellular immunology.

Aim 1 – To complete extensive characterisation of the murine immune response against current panel of purified, refolded recombinant Opa proteins as originally funded by Meningitis UK.

Functional bactericidal antibody titres in immunised versus non-immunised sera against different clonal complexes of meningococci have been performed using the SBA evaluation method. Immunisation with refolded recombinant Opa proteins (rOpaA and rOpaD) elicited bactericidal antibodies against the target N. meningitidis strains only when the target protein was expressed. Other laboratory techniques such as Dot blotting, Western blotting and whole cell ELISA have also been used to measure the level and breadth of antibody responses against rOpa proteins. The characterisation of the murine immune response against recombinant Opa proteins has been completed, providing compelling proof in principle for the development of an Opa-based meningococcal vaccine.

Aim 2 – To systematically investigate the immunomodulatory effects of Opa proteins on proliferation of native human B cells and CD4+ T-cells, on the secretion of pro-inflammatory cytokines from human macrophages and CD4+ T-cells and on the secretion of antibody from B cells.

There are conflicting data in the literature regarding the effect of Opa proteins on CD4+ T-cell proliferation. Through the establishment of several academic collaborations during this project, the team were able to perform experiments using identical reagents to those previously published allowing them to directly compare the results from this study with those in the literature. In contrast to the data presented by Boulton and Gray-Owen (2002)[1] and Lee et al (2007)[2], in these experiments purified recombinant Opa proteins and Opa containing liposomes revealed no consistent stimulatory/inhibitory influence on CD4+ T-cell proliferation or IFN-gamma cytokine production. Inhibition of T-cell proliferation was evident upon co-culture with liposomes containing OpaD although no effect was seen with the purified rOpaD. These experiments were performed on a limited number of individuals (13 participants) and will be extended for publication with several different batches of recombinant protein and new liposome preparations. The team will also investigate whether liposomal aggregation in this particular IOpaD stock preparation was responsible for the results that were obtained.

The data are in line with a publication by van der Ley and Virji in 2009[3], who showed that Opa-/Opa+ OMVs and Opa-/Opa+ meningococci/gonococci induce sustained proliferative responses in CD4+ T-cells. There was no consistent effect of any of the Opa antigens on CD4+ T-cell proliferation which suggests that these cells were able to divide and expand in number following contact with Opa antigens. Antibody production from B cells was also studied but no Opa-mediated inhibition or stimulation was found. No inhibitory effects of Opa proteins were discovered using ex vivo T-cells, which is the standard method of vaccine evaluation. This suggests that any response to an Opa based vaccine should not impact on the development of the immune response. The majority of antigens did not cause a significant response in the healthy volunteers that were studied.

The data provide no evidence that Opa should be excluded from future meningococcal vaccine formulations as they may not necessarily inhibit T-cell function. However, the responses to the Opa antigen panel varied between individuals and this should be taken into consideration when optimising the final vaccine formulation.

Aim 3 – To evaluate the vaccine candidacy of non-native forms of Opa proteins (unfolded recombinant proteins and in vitro manufactured peptides) which could be more advantageous in manufacture and regulation and to explore their effects on aspects of cellular immunology.

In the first year of the study a panel of mutated Opa proteins (non-native) were successfully manufactured. In the second and final year of the study the team evaluated the vaccine candidacy of several non-native forms of Opa protein including unfolded forms of rOpaA and rOpaD (which was achieved using protein denaturing conditions), folded forms of rOpaA mutant and rOpaD mutant proteins and in vitro manufactured Opa peptides. The immunomodulatory effects of non-native forms of Opa were investigated upon co-culture with human PBMCs and CD4+ T-cells.

Immunisation with rOpaD mutant protein elicited SBA titres of 1:256 against target strains expressing this particular Opa protein, however, the rOpaA mutant protein elicited a lower titre against the OpaAD+ target strain compared to the native rOpaA protein. Preliminary results using these protein preparations did not indicate any non-native Opa-mediated effects on T-cell proliferation, T-cell cytokine production or B cell antibody production under the culture conditions studied. This series of experiments was performed with samples from a limited number of study participants and will therefore be extended and included in any future publication.

Immunisation with Opa peptides did not elicit bactericidal antibodies in mice therefore priority for the immunomodulatory assays was given to the protein panel described above. At present, there is no murine SBA data to present for the denatured native and non-native forms of rOpaA and rOpaD but the team hope to determine these titres within the next few months.

Since several of the native and non-native forms of Opa proteins did not influence a number of studied immunomodulatory functions these non-native antigens would be advantageous in the manufacturing of a vaccine formulation.