Genetically engineered N. lactamica as a vaccine delivery agent

One of the main causes of meningitis is the bacterium Neisseria meningitidis (Nm, the meningococcus). Meningococcal disease is a major public health burden worldwide, particularly striking small children and teenagers. The need for improved Nm vaccines is therefore compelling. In this context, the closely related but non-pathogenic species Neisseria lactamica (Nla) which normally resides in the oropharyngeal mucosa is an attractive human mucosal vaccine delivery vector. Modification of Nla to express an optimal profile of Nm-specific and other antigens, relevant for vaccine purposes, is dependent on efficient genetic manipulation. A major obstacle to using Nla in antigen delivery is, however, its recalcitrance to genetic manipulation. The main goal of the VACLAC project was therefore to exploit Nla as a natural vaccine candidate against Nm and to genetically modify Nla to enhance its vaccine potential. 1. Neisseria lactamica reverse genetics to make Nla genetically tractable Nla is very restrictive to DNA uptake and integration. Therefore, we established a novel reverse genetics approach and made Nla amenable to genetic manipulation by knocking out key restriction modification (RM) genes. First, we searched Nla genome sequences for unique RM systems. We also discovered that the 4-nucleotide motif CATG was significantly avoided in all Nla genome sequences. NlaIII is the restriction endonuclease in Nla that recognizes the sequence CATG, and is uniquely present in Nla genome sequences, but not in Nm. We hypothesized that NlaIII-mediated restriction is a major component in the Nla gene transfer barrier. To test this hypothesis, we synthesized a gene knock-out element that lacked any recognition sites for all the four RM systems in Nla. Using this element, we knocked out the gene encoding the NlaIIIR restriction endonuclease. With NlaIIIR gone, we could genetically manipulate Nla. 2. Engineering of Nla strains to express identified Nm vaccine antigens In order to select appropriate potential vaccine antigens, we explored the antigenic and proteomic profile of Nla and Nm cells. A pipeline for enriching neisserial proteins was established, coupled with high-sensitivity mass spectrometry (MS). Thereby, >2100 Nla and Nm proteins were identified. Despite their close relationship, Nla and Nm strains exhibited differentially abundant (DA) protein profiles. The DA proteins between Nla and Nm were mainly secretion components and carbohydrate metabolism enzymes (more abundant in Nla) and iron regulation related (less abundant in Nla). Notably, there was conserved expression of surface exposed outer membrane proteins that are potential vaccine candidates. The Nla and Nm proteomes were used as a basis for selection of potential vaccine candidates and Nla variants to be engineered. Thereby, we could select candidate antigens and engineer Nla to express selected Nm antigens including Opa and FetA. 3. Purification of OMVs from engineered Nla strains and assessing immunoprotectivity Neisserial outer membrane vesicles (OMVs) are attractive for vaccine purposes. Human carriers of Nla develop mucosal and systemic humoral immunity to Nla together with cross-reacting antibodies against Nm. In order to address Nla OMV antigenicity, intranasal and intrathecal administration of Nla and Nm OMVs was performed in a mouse model. By intranasal inoculation, OMVs from an engineered Nla strain were as immunogenic as OMVs from Nm. Further, Nla antigen immunogenicity was assessed by screening human sera for their potential to trigger bactericidial and opsonophagocytic antibodies, yielding PilQ reactivity. 4. 3D structural characterization of targeted neisserial outer membrane-associated antigens and use of this information to engineer Mc antigen display by Nla 3D structural characterization of Nla PilQ, FetA, NadA and Opa was performed. Using a combination of 3D cryoEM and NMR, we examined the membrane-spanning portion of the Nla PilQ multimer, which appeared to harbour potential protective antigens which are likely to be of use in a vaccine. In order to define the immunogenic potential of Nla OMV membrane proteins, a chip with Nla antigen display was manufactured and reactivity with patient sera was tested, yielding the surface-exposed parts of PilQ. 5. Testing of Nla protein glycosylation through reverse genetics and immunoscreens O-linked neisserial protein glycosylation is important for cellular adherence. We detected the Nla protein glycosylation (pgl) gene content and the corresponding oligosaccharide structure. By MS, a new Nla tetrasaccharide glycoform was discovered which potentially is important for protein antigenicity. The VACLAC project builds on interdisciplinary technology in neisserial biology in a translational approach, providing capacity building and training in Norway, UK and Ethiopia. Furthermore, the strategy pursued also represents a model system which can be used for combatting a multitude of infections.

The LACVAC project addresses meningococcal disease which is a major public health burden worldwide, particularly striking small children and teenagers. Virulent isolates of the causative agent, Neisseria meningitidis (the meningococcus, Mc), are covered b y an antiphagocytic capsule. Capsule-based vaccines are available for most Mc strains, but cannot be used for those belonging to serogroup B. The hyperdynamic Mc genome also gives rise to new variants and new serogroups are emerging. The need for new and better vaccines against Mc, including MenA, MenB and MenW, is therefore compelling. Moreover, antigen delivery at mucosal sites has been a major goal in preventing disease many important human infectious diseases. Mucosal immunization can induce both humo ral and cell-mediated protection, at the mucosa as well as systemically. In these contexts, the closely related but non-pathogenic species N. lactamica (Nla) is attractive and promising as a human mucosal vaccine delivery vector. We will genetically modif y the natural Nla candidate to enhance its vaccine potential when delivered intranasally in humans. We will also characterize and test already identified Mc vaccine candidates in Mc and Nla outer membrane vesicles (OMVs), where targeting complex membrane proteins represents a new approach to the search for components with vaccine antigenic potential. The strategy proposed builds on the seminal and complementary findings by our consortium in this field, ranging from basic genetics and antigen discovery to vaccine delivery in a translational approach, highly relevant for capacity building in Norway and LMIC countries and the current GLOBVAC call. Furthermore, the strategy proposed also represents a model system for combating multidrug-resistant gonorrhea an d a multitude of other infectious diseases worldwide.