Process for producing native outer membrane vesicles (NOMVS) from bacteria

Abstract

Disclosed herein is a combination vaccine based on outer membrane vesicles (OMVs) with overexpressed recombinant antigens that is broadly protective against Neisseria meningitidis (Nm) and Neisseria gonorrhoeae (Ng). The OMVs were produced from strains with the acyl transferase LpxL1 knocked out to attenuate endotoxin activity. Factor H binding protein (FHbp) mutants with reduced Factor H (FH) binding from subfamilies A and B were overexpressed in the Nm strains. The Ng strain also had reduction modifiable protein knocked out. The combination vaccine maintained broad serum bactericidal activity (SBA) with human complement against diverse Nm and Ng strains grown in the presence of CMP-NANA, showing no significant reduction in SBA compared to the respective individual vaccines even though the dose of each was decreased by half. Further disclosed are processes for producing outer membrane vesicles (OMVs) from bacteria that acquire iron from human transferrin comprising growing the bacteria in culture medium comprising recombinant human transferrin; compositions comprising outer membrane vesicles (OMVs), bacteria that acquire iron from human transferrin, and recombinant human transferrin.

Description

Attorney Docket No. OMV0005-401-PCPROCESS FOR PRODUCING NATIVE OUTER MEMBRANE VESICEES (NOMVS)FROM BACTERIA

[0001] This application claims the benefit of priority of United States provisional application no. 63 / 777,488, filed March 25, 2025, the contents of which are incorporated by reference as if written herein in their entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Number AI046464 and Grant Number AI124759 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] The present disclosure relates to outer membrane vesicles (OMVs) that have been modified to be effective in the treatment or prevention of disease caused by Neisseria meningitidis or Neisseria gonorrhoeae infections. Further disclosed arc methods of using the OMVs in the treatment or prevention of disease, and methods of making the OMVs.

[0004] Neisseria meningitidis (meningococcus, Nm) is an important cause of sepsis and meningitis worldwide (MacNeil et al. 2018). The organism can be subdivided into capsular groups based on chemically and immunologically distinctive capsular polysaccharides. Strains from five serogroups, designated A, B, C, W, X and Y, are responsible for nearly all cases of invasive meningococcal disease (IMD), and MenB, in particular, causes most disease in infants and has been responsible for most recent outbreaks. Highly effective polysaccharide-protein conjugate vaccines are available against A, C, Y and W strains but not against B, because the B capsule is poorly immunogenic and antibodies to it cross-react with host antigens.

[0005] Meningococcal disease is a rare but dangerous disease that can cause death or severe complications within hours including impaired mental function, deafness, limb loss, and organ failure. Anyone can get meningococcal disease although certain groups such as infants, children, adolescents, and young adults are at increased risk. It is best prevented by vaccination rather than treatment after it occurs because disease progression can be rapid. MenB strains are responsible for approximately one-third of cases of meningococcal disease in the U. S, and up to 80% in Europe.

[0006] Two MenB vaccines that target protein antigens have been licensed in the U. S. and in other countries. One vaccine is called 4CMenB (Bexsero®, GSK) because it contains four components: three recombinant proteins, one of which is Factor H binding protein (FHbp),Attorney Docket No. OMV0005-401-PCand detergent-extracted outer membrane vesicles (dOMV). The second vaccine is called MenB-fHbp (Trumenba®, Pfizer) because it contains two recombinant, lipidated FHbp antigens. MenB-fHbp is approved for individuals > 10 years and 4CMenB is approved for infants as young 2 months of age in Canada. In the United Kingdom, 4CMenB is recommended routinely in the first year of life.

[0007] In 2020, WHO estimated 82.4 million new infections with Neisseria gonorrhoeae (Ng) among adults aged 15 to 49 years. The number of cases of Ng disease in the U. S. has increased by 67% between 2013 and 2018 and another 11% from 2018 to 2020 (from CDC). Prevalence of gonorrhea is highest among vulnerable populations such as men who have sex with men, sex workers, transgender women and adolescents, and young people in high burden countries. In women, Ng infections most frequently occur as cervicitis or pelvic inflammatory disease, which can lead to infertility. About half of infected women have minimal of no clinical manifestations of infection and remain untreated, which leads to further spread of disease. Infants bom to infected mothers can develop ophthalmia neonatorum, which, if untreated, can cause blindness. In men, most Ng infections are manifested as urethritis. Ng also causes anorectal infections, particularly in men who have sex with men. In some cases, Ng infections can develop into disseminated infections with bacteremia leading to arthritis, endocarditis or meningitis. Gonorrhea is treatable and can be cured with antibiotics. However, the emergence of Ng resistant to multiple antibiotics are making treatment of gonorrhea more and more challenging, with the risk of emergence of universally resistant strains and infection becoming untreatable. Despite more than 25 years of work, there is no licensed vaccine against Ng. All of these facets emphasize a significant global public health problem and need for an effective Ng vaccine that can overcome barriers to acceptance.

[0008] Ng infections do not elicit broad protective immunity, which can result in persons developing multiple reinfections. Ng have multiple mechanisms to evade immune clearance including recruiting complement factors FH and C4b binding protein (C4BP) and suppression of cellular functions. Key Ng factors in these mechanisms include porin PorB, NspA, LOS derivatives and OpaD, Opa54, and Opa60 (collectively OpacEA) proteins that bind to CEACAM1 and suppress T cell responses. Because of the varied immune suppression mechanisms, Ng vaccine approaches based on killed Ng bacteria, Ng dOMV or pili have not been successful. While progress has been made with several Ng recombinant protein antigens, including adhesin complex protein (AGP), methionine binding protein MetQ, nutrient acquisition proteins Tfbp, TdfH and TdfJ, and other antigens discovered byAttorney Docket No. OMV0005-401-PCproteomic strategies, as well as truncated LOS, none of these approaches has proven to be broadly protective.

[0009] FHbp (previously referred to as genome-derived antigen (GNA) 1870 or LP2086, is a lipoprotein that is exposed on the surface of meningococci and specifically binds human complement Factor H (FH), which is a down regulator of the alternative complement pathway. Binding of complement FH to the bacterial surface is a critical mechanism by which by N. meningitidis evades innate host defenses. Antibodies to FHbp elicit complement-mediated serum bactericidal activity (SBA), which confers protection against developing invasive meningococcal disease (IMD). Based on amino acid sequence relatedness and immunologic cross-reactivity, FHbp sequence variants can be subdivided into two families, A and B, or three variant groups. Variant group 1 is equivalent to sub-family B and variant groups 2 and 3, to sub-family A. Each amino acid sequence variant is assigned an individual “peptide” identification number or ID, which is available on a public database (https: / / pubmlst.org / ncisscria / fHbp / ). In general, anti-FHbp antibodies elicited by a subfamily A FHbp provide SBA protection against strains with sub-family A variants but not B, and vice versa.

[0010] While the currently licensed MenB vaccines are generally considered safe and effective, their uptake in has been limited. The current ACIP recommendations for 4CMenB or MenB-fHbp are considered on an individual basis depending on strain susceptibility, individual preferences and regional MenB epidemiology for healthy individuals 10 years of age or older in the U. S. In contrast, school-based immunization programs for the quadrivalent meningococcal A, C, Y, W conjugate vaccines resulted in >88% vaccine uptake in the U. S. Contributing to the lack of a general recommendation for MenB vaccines are concerns about incomplete MenB strain coverage (based on susceptibility to SBA, 4CMenB strain coverage in the U. S. recently was estimated to be 67%). Also of concern is the relatively short persistence of SBA after MenB vaccination, and minimal if any protection against nasopharyngeal carriage. In contrast, polysaccharide-protein conjugate vaccines decrease carriage, which extends indirect protection to the community beyond those being vaccinated, and increases cost-effectiveness. Finally, there is a theoretical risk of eliciting autoantibodies in humans to FH by MenB vaccines containing wild-type FHbp that can bind human FH. At the time of the ACIP recommendations, it was not known that FHbp can complex with human FI I, and that anti-FH autoantibodies had been observed in human FI I transgenic mice immunized with 4CMenB. Recent studies confirm that 4CMenB elicits serum anti-FH autoantibodies in infant macaques and adult humans. Serum anti-FH autoantibodies can beAttorney Docket No. OMV0005-401-PCfound naturally in a small proportion of healthy persons and, in most cases, do not appear to have deleterious effects. However, depending on the FH binding specificity, anti-FH autoantibodies can decrease FH function, which is implicated in the pathogenesis of certain human diseases involving complement dysregulation such as autoimmune atypical hemolytic uremic syndrome (aHUS) and complement C3 glomerulopathy (C3G). Although the anti-FH autoantibodies elicited by 4CMenB did not appear to decrease FH function, additional studies are warranted.

[0011] Native outer membrane vesicles (NOMVs) have been proposed as a potential delivery mechanism for vaccines. NOMVs are small, spherical structures that are naturally released by certain types of bacteria and can be isolated and purified for use in vaccines. They are rich in antigens and can be used to present these antigens to the immune system, which can help to stimulate an immune response. Additionally, NOMVs have been shown to have adjuvant properties, which means that they can enhance the effectiveness of the vaccine. However, more research is needed to fully understand the potential of NOMVs as a vaccine delivery mechanism and to develop safe and effective NOMV-based vaccines.[0012| NOMVs are blebbed naturally from (Nm and Ng) bacteria. Previously, vaccine strains have been genetically modified to (a) overexpress Factor H binding protein (FHbp), which is normally present in low abundance, (b) express mutant FHbp with low binding to host Factor H to increase antibody responses that block the interactions causing FH binding, and (c) have attenuated endotoxin by knocking out the acyl transferase, LpxLl, enabling use of NOMV without the detergent treatment that is normally used to decrease reactogenicity, but also results in removal or alteration of potentially protective antigens. The NOMV-FHbp vaccine with penta-acylated lipooligosaccharide (LOS) resulting from knocking out LpxLl decreases cytokine responses in human peripheral blood mononuclear cells (PBMC), which were similar to or lower than those elicited by detergent extracted OMV vaccines that had been safely administered to tens of thousands of human subjects. To further enhance the safety of the NOMV-FHbp vaccine, the strains used to prepare the vaccine incorporate additional genetic deletions that eliminate expression of other undesirable antigens, including the group B capsular polysaccharide, and derivatives of LOS, which are known to cross-react with human glycans having similar structures.

[0013] The immunogenicity of antigens presented in NOMV is greatly increased versus comparable amounts of the recombinant protein alone. However, the most effective antibody responses require a threshold level of expression that has been achieved by using promotersAttorney Docket No. OMV0005-401-PCengineered to produce high rates of transcription, inserting multiple copies in the bacterial genome, and transformation with a multi-copy plasmid.

[0014] The NOMV platform also has adjuvant properties that enhance antibody responses. Overall, NOMV-based vaccines elicit higher titers of antibodies with broader reactivity than the corresponding recombinant proteins and may be more tolerable, since less protein may be required to provide an effective protective antibody response.

[0015] To make NOMVs in amounts that are sufficient for a commercial product, the bacteria must be grown to high densities at which the number of bacteria are high and NOMV production is greatest. Feme iron (Fe3+) is required for bacterial growth.

[0016] Obligate human pathogens, which are microorganisms that can only infect humans, have evolved various mechanisms to acquire iron from host iron-binding proteins. These mechanisms include:a. Siderophore-mediated iron acquisition: Many obligate human pathogens produce small, iron-chelating molecules called siderophores, which can bind and transport iron from host iron-binding proteins.b. Direct binding to host iron-binding proteins: Some obligate human pathogens have surface receptors that can directly bind to host iron-binding proteins such as transferrin, lactoferrin, and haptoglobin.c. Enzyme-mediated degradation of host iron-binding proteins: Some pathogens have enzymes, such as lactoperoxidase or lysozyme, that can degrade host iron-binding proteins, releasing iron in the process.d. Iron uptake from heme-containing molecules: Obligate human pathogens like Neisseria meningitidis and Haemophilus influenzae have the ability to use heme as a source of iron, by taking up and breaking down host hemoglobin or myoglobin molecules.e. Iron uptake from host cells: Some obligate human pathogens, such as Mycobacterium tuberculosis, can enter and grow inside host cells and take up iron from the host cell's iron storage molecules.

[0017] Often the mechanisms are highly specific for the human versions of the proteins and versions from other animal species cannot support growth of the bacteria. Chelated Fe3+in the form of ferric citrate, EDTA, pyrophosphate, or methylenediphosphonic acid can also serve as sources of iron but limited solubility and formation of iron oxides in the presence of oxygen and near neutral pH needed to grow bacteria limits the availability of iron from these sources and the bacterial densities that can be produced.Attorney Docket No. OMV0005-401-PC

[0018] Human sources of iron binding proteins cannot be used for commercial manufacturing of NOMVs because of limited availability and risk of transmitting disease. Alternatively, recombinant human iron binding proteins have been produced in yeast Saccharomyc.es cerevisiae). insect cells (Pichia pastoris), and rice (Oryza sativa ).

[0019] Production in plants such as rice is particularly important because of the relatively high yield and thus lower cost of production, and glycosylation of transferrin produced in rice is similar to that of human transferrin. Rice has been used as a model plant for producing human proteins because of its relatively simple genetic makeup and efficient transformation methods. Rice endosperm, the part of the seed that provides nourishment for the developing plant, has been shown to be a particularly good site for producing human proteins because of its high storage protein content.

[0020] Scientists have used different strategies to produce human transferrin in rice, including:a. using the rice endosperm-specific promoters to drive the expression of the human transferrin gene;b. using the rice endosperm-specific storage protein bodies to compartmentalize the human transferrin protein; andc. using the rice endosperm-specific post-translational modification machinery to correctly fold and process the human transferrin protein.

[0021] It has been discovered that Nm and Ng grown in medium supplemented with human transferrin produced in rice results in high densities of bacteria and production of NOMVs. Also, acquiring ferric iron from a natural host source may up regulate bacterial virulence genes where the protein products of those genes end up in NOMVs and are significant antigens for eliciting antibody responses that are protective against meningococcal and gonococcal disease.

[0022] Finally, there are a number of human and animal pathogens that acquire iron through host transferrin. The use of medium with recombinant host-matched transferrin may be useful for commercial scale production of bacteria that acquire iron from transferrin.

[0023] However, more research is needed to fully understand the potential of NOMVs as a vaccine delivery mechanism and to develop safe and effective NOMV-based vaccines. Thus, there exists a need to develop new, safe, efficient methods and processes for producing NOMVs.Attorney Docket No. GMV0005-401-PCSUMMARY

[0024] In an embodiment, an immunogenic composition comprises outer membrane vesicles (OMVs) obtained from mutant strains of Neisseria, comprising:a) OMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of wild type (WT) Factor H binding protein (FHbp) subfamily B, siaD-galE, and IpxLl genes with subfamily A FHbp ID22 mutant comprises one or more of L130R, G133D, and K218N substitutions; andOMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of WT FHbp subfamily B, siaD-galE, and Ipxl J genes with subfamily B FHbp ID9 single mutant with S228R substitution; andb) OMVs from a Neisseria gonorrhoeae parent strain with decreased or knocked-out expression and / or function of the lipid A biosynthesis lauroyl acyltransferase (IpxLl) gene and reduction of expression of the modifiable protein (rmp) gene.

[0025] In an embodiment, the subfamily A FHbp ID22 mutant comprises two or more of L130R, G133D, and K218N substitutions.

[0026] In an embodiment, the subfamily A FHbp ID22 mutant comprises three of L130R, G133D, and K218N substitutions.

[0027] In an embodiment, the substitutions to wild type FHbp produce a modified FHbp that has reduced binding to human Factor H.

[0028] In an embodiment, the OMV contains four copies of the modified genes that results in overexpression of the FHbp.

[0029] In an embodiment, the disruption of IpxLl gene results in penta-acylated lipo-oligosaccharides.

[0030] In an embodiment, the disruption of the siaD-GalE genes eliminates sialic acidcontaining capsular polysaccharides and LOS antigens.[00311 In an embodiment, both H44 / 76 strains comprise a copy of the recombinant FHbp gene inserted into the fhbp gene, the siaD-galE locus, and the IpxLl gene.

[0032] In an embodiment, the H44 / 76 strains further comprise a plasmid with a 4th copy of the recombinant FHbp gene.

[0033] In an embodiment, the ID22 triple mutant strain and ID9 single mutant strain are present in a 70:30 ratio of ID22: ID9.

[0034] In an embodiment, the composition is adsorbed to aluminum hydroxide adjuvant.Attorney Docket No. OMV0005-401-PC

[0035] In an embodiment, the parent strain of b) is chosen from FA1090, WHO F, WHO G, WHO N, WHO M, and MSI 1.

[0036] In an embodiment, the parent strain of b) has the reduction modifiable protein (rmp) gene completely knocked out.

[0037] In an embodiment, the parent strain of b) has the lipid A biosynthesis lauroyl acyltransferase (Ipxl 1) gene knocked out.

[0038] In an embodiment, the first 120 amino acids of the Ipxl 1 gene are present.

[0039] In an embodiment, the parent strain of b) further comprises a knockout of the one or more of the OpaD, Opa54, Opa60, and Opas genes.

[0040] In an embodiment, the composition is administered via intramuscular injection.

[0041] In an embodiment, the composition is not pyrogenic.

[0042] In an embodiment, a method of eliciting a bi-valent antibody response, comprises administration of the immunogenic composition as described herein.

[0043] In an embodiment, the response elicits antibodies specific for the vaccine antigen present in each component as an NOMV-FHbp or NOMV-Ng bi-valent vaccine or when combined as a tetra-valent combination vaccine (NOMV-combo).

[0044] In an embodiment, a method of eliciting serum bactericidal activity (SBA) against diverse MenB and Ng strains and against both diverse meningococcal and gonococcal strains, comprises administration of the immunogenic composition as described herein.

[0045] In an embodiment, a method of inhibiting Neisseria gonorrhoeae and Neisseria meningitidis colonization of cervical and vaginal cells, comprises administration of the immunogenic composition as described herein.

[0046] In an embodiment, a method of vaccinating a patient against Neisseria gonorrhoeae and Neisseria meningitidis infections comprises administration of the immunogenic composition as described herein.

[0047] In an embodiment, a process for producing outer membrane vesicles (OMVs) from bacteria that acquire iron from human transferrin comprising growing the bacteria in culture medium comprising recombinant human transferrin.

[0048] In an embodiment, a process for producing outer membrane vesicles (OMVs) from bacteria that acquire iron from human transferrin comprises:a) growing the bacteria in an atmosphere of ambient air supplemented with 3 % - 5 % CO2;b) isolating individual cultures of bacteria and dispersing the cultures in medium comprising recombinant human transferrin;Attorney Docket No. OMV0005-401-PCc) growing the redispersed cultures at about 37 °C;d) adding fresh medium and further growing the cultures at about 37 °C in an atmosphere of ambient air supplemented with CO2 one or more times; and e) purifying the cultures.

[0049] In an embodiment, a composition comprises outer membrane vesicles (OMVs), bacteria that acquire iron from human transferrin, and recombinant human transferrin.

[0050] In an embodiment, a pharmaceutical composition comprising purified outer membrane vesicles (OMVs) made by the process described herein and a therapeutically acceptable excipient.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 shows the effect of ferric iron supplements in chemically defined medium on the growth of Neisseria meningitidis strain H44 / 76.

[0052] Figure 2 shows the concentration-dependent effect of recombinant human transferrin in chemically defined medium on the growth of Neisseria meningitidis strain H44 / 76.

[0053] Figure 3 compares the growth Neisseria meningitidis strain H44 / 76 in chemically defined medium supplemented with 2g per liter of either recombinant human transferrin or lactoferrin.

[0054] Figure 4 shows the growth rate of Neisseria meningitidis strain H44 / 76 in chemically defined medium supplemented with 1.5g per liter of recombinant human transferrin when grown in a 30 L fermenter that controls, mixing, temperature, dissolved oxygen and CO2, and pH.

[0055] Figure 5 shows the Sephacryl S-400 elution profile of NOMV and proteins in concentrated culture medium from Neisseria meningitidis strain H44 / 76 grown in chemically defined medium supplemented with 1.5g per liter of recombinant human transferrin at a IL scale.

[0056] Figure 6 shows an SDS-PAGE gel of column fractions taken from the size exclusion chromatography (SEC) purification of NOMV from bacterial cell culture supernatant shown in Figure 5. The samples from different purification steps and run in each lane are shown in the table on the right side of the figure. Lanes 8-10 represent purified NOMV (Peak 1) after SEC separation from transferrin and other impurities. TFF, tangential flow filtration.

[0057] Figure 7 depicts the growth of Neisseria gonorrhoeae strain FA1090 in which the LpxLl and Rmp genes have been knocked out, in Gonococcal base liquid medium supplemented with recombinant human transferrin produced in rice.Attorney Docket No. GMV0005-401-PCDETAILED DESCRIPTIONProteins and Vectors

[0058] In some embodiments, the disclosure provides a transferrin protein. “Transferrin” can refer to a transferrin protein or protein-encoding sequence from an animal, such as a mammal, and including a human. The transferrin family of proteins can be sub-divided into several categories according to amino acid sequence, function, and occurrence in nature. These include: (1) Serum transferrin (serotransferrin, siderophilin, or [3-1 -metal -binding globulin), which is the transferrin found in the serum of vertebrates and other biological fluids including cerebrospinal fluid, milk, and semen. Its main biological function lies in its ability to transfer iron between different biological tissues. (2) Lactotransferrin (lactoferrin) is mainly produced by mucosal epithelial cells of mammals and is thus found abundantly in mammalian milk and other secreted fluids, e.g., tears, saliva, and pancreatic juice. It can also be present in specific granules of polymorphonuclear leukocytes. Lactoferrin exhibits variable properties that have antioxidant, antiinflammatory, and antimicrobial activities. Therefore, it plays an important role in body defense against infections. (3) Ovotransferrin (conalbumin) is primarily present in bird and reptile oviduct secretions and their eggs and constitutes about 12-13% of the egg white in birds. It has the same structural protein, but different glycan component, of serum transferrin, as they are derived from the same gene. (4) Melanotransferrin (p97 cell surface protein), which can be found in trace amounts in normal tissues; however, the majority of human melanomas express this protein, and it is one of the first cell surface markers associated with melanomas.

[0059] One exemplary TF is a single-chain glycoprotein of 679 amino acid residues including 38 cysteine residues which are all disulfide bonded. TF consists of two homologous halves, each comprising about 340 amino acid residues and sharing about 40% sequence identity (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003); Hirose, Biosci. Biotechnol. Biochem. 64:1328-1336 (2000); J. Wally, et al., Biometals 20: 249-62 (2007)). The two homologous halves are shown by X-ray crystallography to fold into two distinct globular lobes called N- and C-terminal lobes (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003); Hirose, Biosci. Biotechnol. Biochem. 64:1328-1336 (2000)). Fach lobe comprises two dissimilar domains (N1 and N2 in the N-lobe; Cl and C2 in the C-lobe) separated by a deep cleft, where the iron binding site is located. The iron-binding ligands inAttorney Docket No. GMV0005-401-PCeach lobe are identical, which involves the side chains of an aspartic acid, two tyrosines, a histidine and two oxygen molecules from a synergistic carbonate anion (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003); Hentze, M. U„ et al., Cell 117: 285-97(2004); Hirose, Biosci. Biotechnol. Biochem. 64:1328-1336 (2000); J. Wally, et al., Biometals 20: 249-62 (2007); Q.-Y. He, et al., “Molecular aspects of release of iron from transferrin,” in: D. M. Templeton, (Ed.), Molecular and Cellular Iron Transport, CRC Press, 2002, pp. 95-124). Neisseria species transferrin binding proteins are thought to be highly specific for human transferrin. These residues, being known to be involved in iron binding, likely should be preserved in a variant transferrin.

[0060] When TF is free of iron (apo-TF), both its N- and C-lobes adopt an open conformation through keeping two domains in each lobe well separated for easy access of the feme iron. At the extracellular pH of 7.4, the apo-TF binds one (monoferric TF) or two iron molecules (diferric TF or holo-TF) by the coordination of iron-binding ligands.

[0061] Recombinant transferrin has been produced successfully in transgenic monocot plants. In some embodiments, transferrin can refer to a recombinant transferrin, or a native transferrin (i.e., not produced from a mutated recombinant gene). In some embodiments, a transferrin protein disclosed herein may be a non-glycosylated form or may be a glycosylated form. In some embodiments, a non-glycosylated form of transferrin lacks observable N-linked glycosylation, within the limits of detection by isoelectric focusing, PNGase F digestion and / or MALDI analysis. In some embodiments, glycosylation status makes no reference to or implications about the O-linked glycosylation status of a protein.

[0062] In some embodiments, a transferrin protein useful with the present disclosure is a plant-derived transferrin. As used herein, “plant-derived” refers to a protein expressed in and isolated or extracted from a plant. Appropriate plants may include a monocot plant or a dicot plant. In some embodiments, a plant suitable for expression of transferrin may be a plant from the family Gramineae, including all members of the grass family of which edible varieties are known as cereals, for example wheat {Triticum sps.), rice {Oryza sps.) barley {Hordeum sps.) oats, {Avena sps.) rye Secale sps.), corn (maize, Zea sps..), millet {Pennisettum sps.), and / or triticale, which is a hybrid of wheat {Triticum sps.) and rye {Secale sps.). Recombinant transferrin produced in transgenic monocot plants such as rice {Oryza sativa L.) is disclosed in US Pat. Publ. No. 2016 / 0076048 and Zhang et al. {BMC Biotech 12:92, 2012). In other embodiments, a plant suitable for expression of transferrin may be a plant from the genus Nicotiana, including members N. tabacum and N. rustica. Plant-derived transferrin may be isolated from any appropriate plant tissue, including, but notAttorney Docket No. GMV0005-401-PClimited to, roots, stems, leaves, leaf sheaths, anthers with pollens, grain husks, pistils, immature seeds, and / or mature seeds.

[0063] Transferrin produced in plant species is known in the art, for example including, but not limited to, rice (Zhang et al., Protein Expr Purif74(V)-.69-79, 2010, sold commercially as Optiferrin™ by Invitria) and wheat germ (sold commercially as TF (Human) Recombinant Protein-GST tag by Abnova). Tobacco (Nicotiana sps.) has also been reported as an expression system for transferrin (Brandsma et al., Plant Biotech J 8(4):489-505, 2020), which describes that recombinant human serum transferrin expressed in transgenic tobacco plants reversibly binds iron in vitro, demonstrates bacteriostatic activity, supports cell growth and proliferation in serum-free culture, and retains the capacity to internalize into mammalian cells in vitro, suggesting that plant-derived human transferrin may have many potential applications.

[0064] In some embodiments, a transferrin useful as described herein may be any transferrin known or available in the art. For example, in some embodiments, transferrins exhibit extensive genetic polymorphism, which can be seen by differences in electrophoretic mobility. Transferrins migrate in the 3-globulin region on starch gel electrophoresis (Smithies and Hiller, 1959). Commonly, there is a single iron-binding band called transferrin C, but faster (B variants) and slower (D variants) migrating bands are occasionally present. Of the three main transferrin variant groups (i.e., Tf-B, Tf-C, and Tf-D), Tf-C encompasses 16 distinct subtypes and is the most common phenotype in all populations.

[0065] Transferrin variants from the same species are usually found to be a result of amino acid substitutions. For example, comparative studies on the chymotryptic digests of human transferrin C and Di indicate that an aspartic residue in transferrin C is replaced by a glycine residue in transferrin Da. On the other hand, in a study on equine transferrin D and R, it was found that one aspartic acid and one glutamic acid residue in transferrin D are replaced by two glycine residues in transferrin R.

[0066] In some embodiments, a transferrin useful in accordance with the present disclosure may be any useful transferrin protein described herein, or a portion thereof, that retains its intended biological activity. Variant proteins are also included within the scope of the proteins to be used in the processes and compositions disclosed herein. Thus, variants of the sequences recited herein are also included. Such variants include natural variants generated by somatic mutation, truncated versions (including fragments), and fusions with other peptides. Alternatively, variants may arise due to the degeneracy of the genetic code or may be produced due to errors in transcription or translation.Attorney Docket No. OMV0005-401-PC

[0067] Further variants of the sequences, whether glycosylated or non-glycosylated, having improved affinity or other properties may be obtained using methods known in the art. For example, amino acid substitutions may be used to obtain proteins with further improved affinity (i.e., affinity maturation). Alternatively, codon optimization of the nucleotide sequence may be used to improve the efficiency of translation in expression systems for the production of a transferrin protein as described herein. Further, polynucleotides comprising a sequence optimized for binding specificity or another attribute may be produced by the application of a directed evolution method to any of the nucleic acid sequences encoding proteins disclosed herein.

[0068] In some embodiments sequences disclosed herein may share 80% or more (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%.97%, 98%, 99% or more) sequence identity with the sequences recited herein. For example, in some embodiments, a recombinant human transferrin described herein has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:1. In some embodiments, a recombinant human transferrin described herein comprises a C-lobe which has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:3. In some embodiments, such sequence identity is calculated with regard to the full length of the reference sequence (i.e. the sequence recited in the application).

[0069] In some instances, the percent identity for a protein or peptide described herein can be reached by the deletion of one or more amino acids from the N-temiinus or C-terminus end of the disclosed peptides.

[0070] In some instances, the disclosed proteins or peptides can comprise one or more unnatural amino acids, modified amino acids, or synthetic amino acid analogues. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, cyclopentylalanine, P-alanine, fluoro-amino acids, designer amino acids such as [3-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogues in general. Also included within the scope are peptides that are differentially modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to anAttorney Docket No. OMV0005-401-PCantibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and / or bioactivity of the protein or peptide.Definitions

[0071] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0073] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, “an active agent” refers not only to a single active agent, but also to a combination of two or more different active agents, “a dosage form” refers to a combination of dosage forms, as well as to a single dosage form, and the like.

[0074] The term “immunogenic composition” refers to a substance that can trigger an immune response when introduced into the body. It typically contains specific molecular structures, such as epitopes, that are recognized by the immune system, prompting the activation of immune cells such as T cells and B cells. Immunogenic compounds can be proteins, peptides, polysaccharides, or other macromolecules, and their effectiveness depends on factors like size, complexity, and foreignness to the host organism. These compounds playAttorney Docket No. GMV0005-401-PCa crucial role in vaccine development, allergenicity studies, and understanding immune system disorders. Immunogenic compositions can be vaccines, immunotherapies, and treatments for immune-related conditions.

[0075] NOMVs that are “naturally blebbed” refer to vesicles that are naturally released by bacterial cells as part of their normal physiology. The outer membrane naturally "buds off," forming vesicles without any external intervention. The process is governed by factors like bacterial growth phase, environmental conditions, and the bacterial strain. This is in contrast to non-naturally blebbed OMVs which are typically produced through external manipulation, such as chemical, mechanical, or genetic modifications to induce vesicle formation. These “non-natural” methods might include detergent treatment, ultrasonication, or hyperosmotic stress. Detergent-extracted OMVs and OMVs produced by treating the bacteria with EDTA or other metal chelating agents are other approaches are also being used to generate OMVs and may benefit from the use of transferrin.

[0076] The terms “polypeptide,” “peptide” and “protein” arc used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. The term “human transferrin” is used herein to denote material that is indistinguishable from transferrin derived from a human or which is a variant or fragment thereof. A “variant” of transferrin may include insertions, deletions and / or substitutions, either conservative or non-conservative, where such changes do not substantially alter the useful ligand-binding or immunogenic properties of transferrin.Naturally-occurring polymorphic variants of human transferrin or human transferrin analogues are included. Generally, variants or fragments of human transferrin will have at least 50% (preferably at least 80%, 85, 90%, 95%, or 99%) of human transferrin’s ligandbinding activity (e.g., iron-binding), weight for weight. Transferrin variants useful for the present disclosure would maintain functionality of non- variant forms of transferrin. A number of transferrin variants can be found in the art, e.g., Ali et al., J Biol Chem 274:24066-24073(1999)). Additionally, single or multiple heterologous fusions of any of the above, or a variant or fragment of any of these may be used. Such fusions include transferrin N-terminal fusions, transferrin C-terminal fusions, and co-N-terminal and C-terminal transferrin fusions.

[0077] The term “sequence identity” means nucleic acid or amino acid sequence identity in two or more aligned sequences, aligned using a sequence alignment program.Attorney Docket No. GMV0005-401-PC

[0078] The term “% homology” is used interchangeably herein with the term “% identity” and refers to the level of nucleic acid or amino acid sequence identity between two or more aligned sequences, when aligned using a sequence alignment program. For example, 70% homology means the same thing as 70% sequence identity determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 70% sequence identity over a length of the given sequence. Exemplary levels of sequence identity include, but are not limited to 70%, 75% 80%, 85%, 90% or 95% or more sequence identity to a given sequence, e.g., the coding sequence for transferrin, as described herein.

[0079] As used herein, “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention.

[0080] The transferrin proteins disclosed herein also include all variants thereof, whether allelic variants or synthetic variants. A “variant” transferrin protein-encoding nucleic acid sequence may encode a variant transferrin protein amino acid sequence that is altered by one or more amino acids from the native transferrin protein sequence, for example at least one amino acid substitution, deletion or insertion. The nucleic acid substitution, insertion or deletion leading to the variant may occur at any residue within the sequence, as long as the encoded amino acid sequence maintains substantially the same biological activity of the native transferrin protein. In another embodiment, the variant transferrin protein nucleic acid sequence may encode the same polypeptide as the native sequence but, due to the degeneracy of the genetic code, the variant has a nucleic acid sequence altered by one or more bases from the native polynucleotide sequence.

[0081] The variant amino acid sequence may contain a “conservative” substitution, wherein the substituted amino acid has structural or chemical properties similar to the amino acid which it replaces and physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature (as determined, e.g., by a standard Dayhoff frequency exchange matrix or BLOSUM matrix). In addition, or alternatively, the variant amino acid sequence may contain a “non-conservative” substitution, wherein the substituted amino acid has dissimilar structural or chemical properties to the amino acid it replaces. Standard substitution classes include six classes of amino acids based on common side chain properties and highest frequency of substitution in homologous proteins in nature,Attorney Docket No. OMV0005-401-PCas is generally known to those of skill in the art and may be employed to develop variant transferrin protein-encoding nucleic acid sequences. A transferrin protein-encoding nucleotide sequence may be engineered in order to alter the transferrin protein coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and / or expression of the transferrin protein by a cell.

[0082] The term “OD620nm” refers to the optical density at 620nm of cells, e.g. Neisseria meningitidis (e.g. strain H44 / 76) cultures, in chemically defined medium. The optical density at 620 nm is used to measure bacterial cell concentration because it is a measure of the cloudiness or turbidity of the culture, indicating the concentration of bacteria by how much light is scattered when a light beam passes through it. The absorption of light at this wavelength is proportional to the number of cells present in the solution, thus it can be used as an indirect measure of cell concentration. Measuring the optical density of bacterial cells at 620 nm does not damage or hinder bacterial growth. The measurement is a non-invasive method that only measures the light absorbance of the cells and docs not affect the cells themselves.[00831 A chemically defined medium is a growth medium suitable for the in vitro cell culture of human or animal cells in which all of the chemical components are known

[0084] The term “native outer membrane vesicle” or “NOMV” means vesicle spherical structures generally less that 1 micrometer in diameter, produced naturally by microbial cells during growth in culture medium from the cell envelop and have not been modified by treatment with chemicals or detergents.

[0085] The term “outer membrane vesicle” or “OMV” relates to proteoliposomic vesicles obtained by disruption of, or blebbing from the outer membrane of Gram-negative bacteria, to form vesicles therefrom that retains antigens from the outer membrane. Gram-negative bacteria naturally shed OMVs which are released into the growth medium. Heterologous antigens are expressed in the Gram-negative bacteria such that they assemble in the membrane that is then released into the culture supernatant. OMVs from such bacteria are representative of the outer membrane and periplasmic bacterial compartments and allow the presentation of membrane proteins in their natural composition and structure. In the broadest sense, OMVs relates to any such proteoliposomic vesicles. However, the term OMVs includes ‘Native OMVs’ (NOMVs), microvesicles (MVs), detergent-extracted OMVs (DOMVs), and blebs, which are outer-membrane protrusions that remain attached to bacteria prior to release as MVs.Attorney Docket No. GMV0005-401-PC

[0086] The term “gonococcal recombinant protein” means a recombinant form of a protein from Neisseria gonorrhoeae. The term “gonococcal recombinant lipoprotein” includes such a protein that is modified to be a lipoprotein, for example by eliminating portions of the protein that are not surface exposed and adding a lipoprotein signal sequence to the remaining C-terminal portion, so that the gonococcal recombinant protein is displayed on the surface of the bacteria in which they are produced, and NOMV are produced by the bacteria as lipoproteins.

[0087] As used herein, the term “sequence identity” means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods. Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc.). See generally, Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm.

[0088] As used herein the term “gene deletion” or “gene knockout” refers to a combination of genetic techniques that has the potential to render a specific gene inoperable or inactive. In some embodiments a gene deletion decreases or abolishes expression of a polypeptide from the gene. In some embodiments both the mRNA and protein are reduced or eliminated. In certain embodiments the expression of gene is partially knocked out or completed knocked out. Partially knocked out means that the expression of a gene is reduced by at least 70%, at least 80%, at least 90%, at least 95% or at least 98% when compared to an endogenous level of expression of a gene. In a certain embodiment the expression of a gene is completely knocked out. Completely knocked out means that, using techniques to monitor the expression of either the mRNA transcribed from a gene, or the expression of protein translated from a particular mRNA, no level of detection is observed. Expression of a gene can be determined by a suitable technique (e.g., by measuring transcript levels by RT / Q-PCR or expressed protein levels by immunoassay e.g. Western Blot). Such techniques are known to the person skilled in the art. Gene deletion or gene knockout, might include not only deletion of geneticAttorney Docket No. OMV0005-401-PCelements but also addition, substitution or modification, such that the gene is inoperable or inactive, i.e. insertion of a genetic sequence may cause mistranslation of the gene, by for example, incorporating an early stop codon, or by causing a missense translation. Genes may for example be deleted by replacement of the gene, or a fragment of said gene, with a different heterologous gene (e.g. an antibiotic resistance gene) for example by homologous recombination.

[0089] The formation of non-naturally blebbed NOMVs involves external manipulation of bacterial cells to induce the release of outer membrane vesicles (OMVs). These methods typically involve chemical, physical, or genetic approaches to disrupt the normal balance of membrane tension, integrity, or synthesis. OMVs that are not blebbed can be produced by methods known in the art. These methods include (but are not limited to) the use of chemical agents, physical induction methods, genetic engineering, and combinations thereof.

[0090] The use of chemical agents such as detergents, chelating agents, or enzymatic treatments to destabilize the bacterial outer membrane, promoting vesicle formation.Detergents like Triton X-100, SDS, or bile salts disrupt lipid interactions in the bacterial outer membrane, causing it to fragment and release vesicles. Chelating agents such as EDTA bind divalent cations (e.g., Mg2+, Ca2+) that stabilize lipopolysaccharide (EPS) interactions in the outer membrane, weakening the membrane and promoting vesicle release. Enzymes like lysozyme or proteases can selectively degrade parts of the bacterial cell wall or membrane proteins, creating instability that facilitates vesicle formation. Sublethal concentrations of antibiotics can induce the release of OMVs in bacteria. Antibiotics at sublethal levels impose stress on bacterial cells, altering membrane dynamics and promoting vesiculation as a bacterial survival mechanism.

[0091] Physical induction methods such as sonication, shear stress, and hyperosmotic stress can be applied to bacterial cells to stimulate vesicle formation. Sonication uses high-frequency sound waves disrupt bacterial membranes, fragmenting them into vesicles. High-shear environments, such as those created by ultrafiltration or high-speed stirring, can cause membrane fragmentation and vesicle release. Exposing bacteria to hyperosmotic conditions (e.g., by adding high salt or sugar concentrations) can force the outer membrane to form vesicles as a stress response.

[0092] Genetic modifications to bacterial strains can enhance vesicle production by altering genes involved in membrane integrity, synthesis, or vesicle regulation. Knockouts or mutations in genes encoding proteins which stabilize the outer membrane lead to increased vesicle production. Overexpressing genes that promote vesiculation, such as those involved inAttorney Docket No. OMV0005-401-PClipid synthesis or LPS transport can enhance OMV release. Synthetic biology techniques allow fine-tuning of vesicle production pathways by introducing entirely new regulatory mechanisms, such as inducible promoters controlling vesicle-associated genes.EXAMPLES

[0093] Examples of embodiments of the present disclosure are provided in the following examples. The following examples are presented only by way of illustration and to assist one of ordinary skill in using the disclosure. The examples are not intended in any way to otherwise limit the scope of the disclosure.Example 1

[0094] Knocking out fhbp, siaD-galE, and IpxLl genes by insertion of a gene coding for the FHbp ID9 SM (ID9 with S228R substitution) and FHbp ID22 TM (ID22 with L130R, G133D, and K218N substitutions) antigens in Neisseria meningitidis serogroup B strain H44 / 76.Transformation of N. meningitidis.

[0095] The H44 / 76 strain in which the fhbp, siaD-galE, and IpxLl genes were inactivated (H44 / 76AFHbp ACapsule AlpxLl) and copies of FHbp ID9 SM, and / or FHbp ID22 TM were inserted was made by homologous recombination by transformation with plasmids pBS-FHbpKO-[FHbp ID9 SM or FHbp ID22 TM]-ERM using erythromycin selection (10 pg / ml), pGEM-SiaD / GalEKO-[FHbp ID9 SM or FHbp ID22 TM]-SPC using spectinomycin selection (50 pg / ml), pUC18-lpxLlKO-[FHbp ID9 SM or FHbp ID22 TM]-KAN using kanamycin selection (50 pg / ml), and pFP12-[FHbp ID9 SM or FHbp ID22 TM]-CAT using chloramphenicol selection (5 pg / ml). Transformations starting from the wild-type strain were carried in the following order:i. The capsule genes were knocked out and the first copy of [FHbp ID9 SM or FHbp ID22 TM] was added (pGEM-SiaD / GalEKO-[FHbp ID9 SM or FHbp ID22 TM]- SPC plasmid);ii. The IpxLl gene was knocked out and a second copy of [FHbp ID9 SM or FHbp ID22 TM] was added (pUC18-lpxLlKO-[FHbp ID9 SM or FHbp ID22 TM]- KAN plasmid);iii. The FHbp gene was knocked out and a third copy of [FHbp ID9 SM or FHbp ID22 TM] was added (pBS-FHbpKO-[FHbp ID9 SM or FHbp ID22 TM]-ERM plasmid).Attorney Docket No. OMV0005-401-PCiv. Overexpression of [FHbp ID9 SM or FHbp ID22 TM] (pFP12-[FHbp ID9 SM or FHbp ID22 TM]-CAT plasmid).

[0096] Ten to 15 colonies of the H44 / 76 strain were selected from a TSB (Tryptic Soy Broth, non-animal origin) agar plate that had been grown overnight. The colonies of bacteria were mixed with 1-3 pg of the plasmid, plated onto a TSB agar plate, and incubated for 6 hrs at 37°C. Serial dilutions of the bacteria were re-cultured onto TSB agar plates containing antibiotic for selection. The culture plates were incubated overnight at 37°C, and the colonies were screened for FHbp ID9 SM or FHbp ID22 TM expression and for the lack of expression of native FHbp, Capsule, and IpxLl by a flow cytometry assay using specific antibodies, and by PCR using heat killed cells. Positive individual colonies were frozen in 10% skim milk (wt / vol) and 15% glycerol, and stored at -80°C. The FHbp ID9 SM and FHbp ID22 TM sequences are shown in Table 1. Primers that go into pUC18 Lpxl, pBS FHbp, and pFP12 plasmids are shown in Table 2. Primers that go into pGEM SiaD / GalE plasmid are shown in Table 3. Other exemplary sequences arc shown in Table 4.Table 1.Identifier Protein Sequence MVNRTAFCLSLTAALILTACSSGGGGSGGGGVAAD IGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKL KLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQ SEQ ID FHbp ID9 IEVDGQLITLESGEFQVYKQSHSALTALQTEQVQD NO: 1 SM SEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSAT YRGTAFGSDDAGGKLTYTIDFAVKQGHGKIEHLKS PELNVDLAAAYIKPDKKRHAVISGSVLYNQDEKGSYRLGIFGGQAQEVAGSAEVETANGIHLIGLAAKQAttorney Docket No. GMV0005-401-PCIdentifier Protein Sequence ATGGTGAACCGAACTGCCTTCTGCCTTTCTCTGAC CGCCGCCCTGATTCTGACCGCCTGCAGTAGCGGAG GCGGCGGAAGCGGAGGCGGCGGTGTCGCCGCCGAC ATCGGTGCGGGGCTTGCTGATGCACTAACCGCACC GCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGA CGCTGGATCAGTCCGTCAG G AAAAAC G AG AAAC T G AAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGG AAACGGCGACAGCCTCAATACGGGCAAATTGAAGA ACGACAAGGTCAGCCGCTTCGACTTTATCCGTCAA ATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAG CGGAGAGTTCCAAGTGTACAAACAAAGCCATTCCG SEQ ID FHbp ID9 CCTTAACCGCCCTTCAGACTGAGCAAGTACAAGAC NO: la SM TCGGAGGAITCCGGGAAGATGGTTGCGAAACGCCA GTTCAGAATCGGCGACATAGCGGGCGAACATACGT CTTTTGACAAGCTTCCCAAAGGCGGCAGTGCGACA TATCGCGGGACGGCGTTCGGTTCAGACGATGCTGG C G G AAAAC TGACCTATACTATAGATTTCGCCGTCA AACAGGGACACGGCAAAATCGAACATTTGAAATCG CCCGAACTCAATGTCGACCTGGCCGCCGCCTATAT CAAGCCGGATAAAAAACGCCATGCCGTCATCAGCG G T T C C G T C C T T T AC AAC C AAG AC G AGAAAG G C AG T TACCGCCTCGGCATCTTTGGCGGGCAAGCCCAGGA AGTTGCCGGCAGCGCGGAAGTGGAAACCGCAAACG GCATACACCTTATCGGTCTTGCCGCCAAGCAGTAA MVNRTAFCLSLTAALILTACSSGGGGSGGGGVAAD IGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKL KLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQ SEQ ID FHbp ID22 IEVDGQLITLESGEFQVYKQSHSALTALQTEQVQD NO: 2 TM SEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSAT YRGTAFGSDDAGGKLTYTIDFAVKQGHGKIEHLKS PELNVDLAAAYIKPDKKRHAVISGSVLYNQDEKGS YRLGIFGGQAQEVAGSAEVETANGIHLIGLAAKQ ATGGTGAACCGAACTGCCTTCTGCCTTTCTCTGAC CGCCGCCCTGATTCTGACCGCCTGCAGTAGCGGAG GCGGCGGAAGCGGAGGCGGCGGTGTCGCCGCCGAC ATCGGTGCGGGGCTTGCTGATGCACTAACCGCACC GCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGA C G C T GG AT C AG T C C GT C AGGAAAAAC GAGAAAC T G AAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGG AAACGGCGACAGCCTCAATACGGGCAAATTGAAGA SEQ ID FHbp ID22ACGACAAGGTCAGCCGCTTCGACTTTATCCGTCAA NO: 2a TMATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAG CGGAGAGTTCCAAGTGTACAAACAAAGCCATTCCG CCTTAACCGCCCTTCAGACTGAGCAAGTACAAGAC TCGGAGGATTCCGGGAAGATGGTTGCGAAACGCCA GTTCAGAATCGGCGACATAGCGGGCGAACATACGT CTTTTGACAAGCTTCCCAAAGGCGGCAGTGCGACA TATCGCGGGACGGCGTTCGGTTCAGACGATGCTGGCGGAAAACTGACCTATACTATAGATTTCGCCGTCAAttorney Docket No. GMV0005-401-PCIdentifier Protein Sequence AACAGGGACACGGCAAAATCGAACATTTGAAATCG CCCGAACTCAATGTCGACCTGGCCGCCGCCTATAT CAAGCCGGATAAAAAACGCCATGCCGTCATCAGCG GTTCCGTCCTTTACAACCAAGACGAGAAAGGCAGT TACCGCCTCGGCATCTTTGGCGGGCAAGCCCAGGA AGTTGCCGGCAGCGCGGAAGTGGAAACCGCAAACGGCATACACCTTATCGGTCTTGCCGCCAAGCAGTAATable 2.I dent if ier Protein SequenceFHbp ID9 SMSEQ ID NO: 3forward primer atcaCatatggtgaaccgaactgcc FHbp ID9 SMSEQ ID NO: 4reverse primer agtagtCCTGCAGGAGGCCTTTAC FHbp ID22 TMSEQ ID NO: 5forward primer a C t a C t C AT AT G C G G T AT G TAA AAAAAG FHbp ID22 TMSEQ ID NO: 6reverse primer agtagtCCTGCAGGAGGCCTCTACTable 3.I dont x f XG r Protein SequenceFHbp ID9 SMSEQ ID NO: 7forward primer atacaattgCCTCAGCGCATGCATC FHbp ID9 SMSEQ ID NO: 8reverse primer tattctagaAGGCCTTTACTGCTTGGC3 FHbp ID22 TMSEQ ID NO: 9forward primer atacaattgCCTCAGCGCATGCATC FHbp ID22 TMSEQ ID NO: 10reverse primer tattctagaAGGCCTCTACTGTTTGCCGTable 4.Identifier Protein SequenceAttorney Docket No. GMV0005-401-PCCCTCAGCGCATGCATCCGGGCAGCAGCGCATCGG CTCGCACGAGGTCTGCGCTTGAATTGTGTTGTAG AAACACAACGT T T TT GAAAAAATAAGCTATT GT T T TAT AT C AAAAT AT AAT C AT T T T T AAAAT AAAGG TTGCGGCATTTATCAGATATTTGTTCTGAAATGA AGACGTATCGGGTGTTTGCCCGATGTTTTTAGGT T T T T AT C AAAT T T AC AAAAGG AAGC C CAT AT G GT GAACCGAACTGCCTTCTGCCTTTCTCTGACCGCC GCCCTGATTCTGACCGCCTGCAGTAGCGGAGGCG GCGGAAGCGGAGGCGGCGGTGTCGCCGCCGACAT CGGTGCGGGGCTTGCTGATGCACTAACCGCACCG CTCGACCATAAAGACAAAGGTTTGCAGTCTTTGA C GC TG GAT C AG T C C G T CAG GAAAAAC GAGAAAC T GAAGCTGGCGGCACAAGGTGCGGAAAAAACTTAT GGAAACGGCGACAGCCTCAATACGGGCAAATTGA AGAACGACAAGGTCAGCCGCTTCGACTTTATCCG TCAAATCGAAGTGGACGGGCAGCTCATTACCTTG GAGAGCGGAGAGTTCCAAGTGTACAAACAAAGCC ATTCCGCCTTAACCGCCCTTCAGACTGAGCAAGT ACAAGACTCGGAGGATTCCGGGAAGATGGTTGCG AAACGCCAGTTCAGAATCGGCGACATAGCGGGCG AACATACGTCTTTTGACAAGCTTCCCAAAGGCGG CAGTGCGACATATCGCGGGACGGCGTTCGGTTCA GACGATGCTGGCGGAAAACTGACCTATACTATAG ATTTCGCCGTCAAACAGGGACACGGCAAAATCGA ACATTTGAAATCGCCCGAACTCAATGTCGACCTGBlue script GCCGCCGCCTATATCAAGCCGGATAAAAAACGCCPlasmid (FHbpSEQ ID NO: 11 ATGCCGTCATCAGCGGTTCCGTCCTTTACAACCA KO+FHbp ID9AGACGAGAAAGGCAGTTACCGCCTCGGCATCTTT SM)GGCGGGCAAGCCCAGGAAGTTGCCGGCAGCGCGG AAGTGGAAACCGCAAACGGCATACACCTTATCGG TCTTGCCGCCAAGCAGTAAAGGCCTCCTGCAGGC C AAGCAAAACC AT TGT GAAAAT GCC GT CC GAACA CGATAATTTACCGTTCGGACGGCATTTTGTATTG CACCGTCCGACGGCATGCCCAAGGGGGGAAATCC CTATTTTCAGGCCAACCGCTATATAATGCCGTCT GAACCAACGAGAGAATGCCATGCAAGCTGATTTT AACCGTCCCGTCCTGGCCGTCGATACCGGTACTT CCCGTTTGTCGCTCGCGCTGCGTGCCGACGGCGA AACCCGTCTGTTCCATCAGGAAGTCGGCAGCCGC CAGTCCGAACTGATTCTGCCGGAAATCCGCACCC TATTCCGCGATGCAGGCATTACCGCCGCCGATTT GGGTGCGGTCGTGTACGCACAGGGTCCCGGCGCG TTTACCGGACTGCGTATCGGCATCGGTGTAGCTC AGGGTTTGGCAACGCCGTTTGATACCCCCTTAAT CGGCGTACCCTCGCTCGATGCCGCCGCCTCGCTG CCGCCGCCGCAAAGCTGCATCCTTGCCGCTACGG ACGCTCGTATGGGCGAAGTGTTTTATGCATGGTT CGATACGCTGCTCGAGGGGGGGCCCGGTACCAGC TTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCT TGGCGTAATCATGGTCATAGCTGTTTCCTGTGTG AAATTGTTATCCGCTCACAATTCCACACAACATA CGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTG CCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGAttorney Docket No. GMV0005-401-PCTCGTGCCAGCTGCATTAATGAATCGGCCAACGCG CGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTC GTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA AGGCGGTAATACGGTTATCCACAGAATCAGGGGA TAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG C AAAAGGC C AG G AAC C GT AAAAAGG C C GC GT T GC TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA GCATCACAAAAATCGACGCTCAAGTCAGAGGTGG CGAAACCCGACAGGACTATAAAGATACCAGGCGT TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGT TCCGACCCTGCCGCTTACCGGATACCTGTCCGCC TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATA GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC CCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGA CTTATCGCCACTGGCAGCAGCCACTGGTAACAGG ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG AGTTCTTGAAGTGGTGGCCTAACTACGGCTACAC TAGAAGAACAGTATTTGGTATCTGCGCTCTGCTG AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT CTTGATCCGGCAAACAAACCACCGCTGGTAGCGG TGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGC AGAAAAAAAGG AT C T CAAGAAG AT C C T T T GAT C T TTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCA AAAAGGAT C T T C AC C T AG AT C C T T T T AAAT T AAA AAT GAAGT T TT AAAT CAAT CT AAAGT AT ATAT GA GTAAACTTGGTCTGACAGTTACCAATGCTTAATC AGTGAGGCACCTATCTCAGCGATCTGTCTATTTC GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA GATAACTACGATACGGGAGGGCTTACCATCTGGC CCCAGTGCTGCAATGATACCGCGAGACCCACGCT CACCGGCTCCAGATTTATCAGCAATAAACCAGCC AGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCA ACTTTATCCGCCTCCATCCAGTCTATTAATTGTT GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAA TAGTTTGCGCAACGTTGTTGCCATTGCTACAGGC ATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTT CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGT TACATGATCCCCCATGTTGTGCAAAAAAGCGGTT AGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA AGTTGGCCGCAGTGTTATCACTCATGGTTATGGC AGCACTGCATAATTCTCTTACTGTCATGCCATCC GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACC GAGTTGCTCTTGCCCGGCGTCAATACGGGATAAT ACCGCGCCACATAGCAGAACTTTAAAAGTGCTCA TCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC AAGGATCTTACCGCTGTTGAGATCCAGTTCGATG TAACCCACTCGTGCACCCAACTGATCTTCAGCAT CTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTAttorney Docket No. GMV0005-401-PCTCCTTTTTCAATATTATTGAAGCATTTATCAGGG TTATTGTCTCATGAGCGGATACATATTTGAATGT ATTTAGAAAAATAAACAAATAGGGGTTCCGCGCA C AT T T C C C C GAAAAG T GC C AC C T AAAT T G T AAGC GTTAATATTTTGTTAAAATTCGCGTTAAATTTTT GTTAAATCAGCTCATTTTTTAACCAATAGGCCGA AAT C G GC AAAAT C C C T T AT AAAT C AAAAGAAT AG ACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGA ACAAGAGTCCACTATTAAAGAACGTGGACTCCAA C GT C AAAG GGC G AAAAAC C GT C T AT C AGG GC G AT GGCCCACTACGTGAACCATCACCCTAATCAAGTT TTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCG GAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGA CGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAG GGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCT GGCAAGTGTAGCGGTCACGCTGCGCGTAACCACC ACACCCGCCGCGCTTAATGCGCCGCTACAGGGCG CGTCCCATTCGCCATTCAGGCTGCGCAACTGTTG GGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTA CGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTC ACGACGTTGTAAAACGACGGCCAGTGAATTGTAA TACGACTCACTATAGGGCGAATTGGAGCTCCACC GCGGTGGCGGCCGCTCTAGACCAGCCACGGCGCA TACAAATTCACCCGTCCGCCCACAGGCGATGTAT TACGTATCGACCGCATCAAAGAAATCCACCAAGC CCTGCCCAATACACACATCGTGATGCACGGCTCC AGCTCCGTTCCGCAAGAATGGCTGAAAGTCATCA ACGAATACGGCGGCAATATCGGCGAAACCTACGG CGTGCCGGTTGAAGAAATCGTCGAAGGCATCAAA CACGGCGTGCGCAAAGTCAACATCGATACCGACT TGCGCCTTGCTTCTACCGGCGCGGTACGCCGCTA CCTTGCCGAAAATCCGTCCGACTTTGACCCGCGC AAAT AC C T GAG C AAAACC AT T GAGG C C AT GAAGC AAATCTGCCTCGACCGTTATCTTGCGTTTGGCTG CGAAGGTCAGGCAGGCAAAATCAAACCTGTTTCG TTGGAAAAAATGGCAAGCCGTTATGCCAAGGGCG AAT T GAAC CAAAT C GT CAAAT AACAGGT T GC C T G TAAACAAAATGCCGTCCCATGGGCAAACTTAAGA GTGTGTTGATAGTGCAGTATCTTAAAATTTTGTG TATAATAGGAATTGAAGTTAAATTAGATGCTAAA AATTTGTAATTAAGAAGGAGGGATTCGTCATGTT GGTATTCCAAATGCGTAATGTAGATAAAACATCT ACTGTTTTGAAACAGACTAAAAACAGTGATTACG C AGATAAAT AAAT AC GTT AGAT T AAT T CC TAG CA GTGACTAATCTTATGACTTTTTAAACAGATAACT AAAAT TACAAA CAAAT CGT T TAACT T CTGTAT T T ATTTACAGATGTAATCACTTCAGGAGTAATTACA T GAAC AAAAAT AT AAAAT AT T C T CAAAAC T T T T T AACGAGTGAAAAAGTACTCAACCAAATAATAAAA CAATTGAATTTAAAAGAAACCGATACCGTTTACG AAATTGGAACAGGTAAAGGGCATTTAACGACGAA ACTGGCTAAAATAAGTAAACAGGTAACGTCTATT GAATTAGACAGTCATCTATTCAACTTATCGTCAGAAAAAT TAAAACT GAACAT T CGT GT CACT TT AATAttorney Docket No. GMV0005-401-PCTCACCAAGATATTCTACAGTTTCAATTCCCTAAC AAACAGAGGTATAAAATTGTTGGGAGTATTCCTT ACCATTTAAGCACACAAATTATTAAAAAAGTGGT TTTTGAAAGCCATGCGTCTGACATCTATCTGATT GTTGAAGAAGGATTCTACAAGCGTACCTTGGATA TTCACCGAACACTAGGGTTGCTCTTGCACACTCA AGTCTCGATTCAGCAATTGCTTAAGCTGCCAGCG GAATGCTTTCATCCTAAACCAAAAGTAAACAGTG TCTTAATAAAACTTACCCGCCATACCACAGATGT TCCAGATAAATATTGGAAGCTATATACGTACTTT GTTTCAAAATGGGTCAATCGAGAATATCGTCAAC T GT T T AC T AAAAAT C AGT T T C AT CAAGC AAT GAA ACACGCCAAAGTAAACAATTTAAGTACCATTACT TAT G AGO AAGT AT TGTCTATTTT T AAT AGT TAT 0 TATTATTTAACGGGAGGAAATAATTCTATGAGTC GCTTTTTTAAATTTGGAAAGTTACACGTTACTAA AGGGAATGGAGATAAATTATTAGATATACTACTG ACAGCTTCCAAGAAGCTAAAGAGGTCCCTTTCGA CGGCCCCGGGFHbpID9SEQ ID NO: 12 DownstreamForward GAAATGAAGACGTATCGGGTGFHbpID9SEQ ID NO: 13 DownstreamReverse ATGGTGTATGCCGTTTGCGGFHbpID9SEQ ID NO: 14 UpstreamForward GGCGAAATCGGCGTATTGGGFHbpID9SEQ ID NO: 15 UpstreamReverse CTACATTACGCATT T GGAAT AC CAttorney Docket No. GMV0005-401-PCGGGCGAATTCCTGGACACGGCAGACCGATAAAGC CCGCACCGCCGGTAACGAGGATGGTTTTTTTGTT TGCGGTTTGCATTTAATCCCCATATCCGTTGGGG TTGCGGCTGACCCAACGCCACGAATCTTCCATCA TTTGCTGCAAGCCGCGTTTGGTTTCCCAGCCGGT TTGTTGTTTGGTATGGGACGGGTCGGCAAAGGAA CACGCCAAATCACCGGCGCGGCGCGGTTTGATTT CAAATGGAATGGTTAAACCCGAAGCCGCTTCAAA TGCGCGGATGATTTCCAATACCGAAGAAGCGCGG CCGGAGCCTAAGTTCAGCAAATGCGTGCCTGCTA CATTACTTTTTGCCTGCATAGCCGCGACATGGCC TTCTGCCAAATCCATCACATGAATATAGTCACGC ATCCCCGTGCCGTCGGGGGTAGGGTAGTCATCGC CAAATACCGCCAATTGCCTCAGCGCATGCATCCG GGCAGCAGCGCATCGGCTCGCACGAGGTCTGCGC TTGAATTGTGTTGTAGAAACACAACGTTTTTGAA AAAAT AAGC T AT TGTTTTATAT CAAAAT AT AAT CATTTTTAAAATAAAGGTTGCGGCATTTATCAGATATTTGTTCTGAAATGAAGACGTATCGGGTGTTTG CCCGATGTTTTTAGGTTTTTATCAAATTTACAAA AGGAAGCCCATATGGTGAACCGAACTGCCTTCTG CCTTTCTCTGACCGCCGCCCTGATTCTGACCGCC TGCAGTAGCGGAGGCGGCGGAAGCGGAGGCGGCG GTGTCGCCGCCGACATCGGTGCGGGGCTTGCTGA T GC AC T AAC CG C AC C GCT C GAC C AT AAAG AC AAA GGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCApGEM Plasmid GGAAAAACGAGAAACTGAAGCTGGCGGCACAAGG(CapsuleSEQ ID NO: 16 TGCGGAAAAAACTTATGGAAACGGCGACAGCCTC KO+FHbp ID9AATACGGGCAAATTGAAGAACGACAAGGTCAGCC SM)GCTTCGACTTTATCCGTCAAATCGAAGTGGACGG GCAGCTCATTACCTTGGAGAGCGGAGAGTTCCAA GTGTACAAACAAAGCCATTCCGCCTTAACCGCCC TTCAGACTGAGCAAGTACAAGACTCGGAGGATTC CGGGAAGATGGTTGCGAAACGCCAGTTCAGAATC GGCGACATAGCGGGCGAACATACGTCTTTTGACA AGCTTCCCAAAGGCGGCAGTGCGACATATCGCGG GACGGCGTTCGGTTCAGACGATGCTGGCGGAAAA CTGACCTATACTATAGATTTCGCCGTCAAACAGG GACACGGCAAAATCGAACATTTGAAATCGCCCGA ACTCAATGTCGACCTGGCCGCCGCCTATATCAAG CCGGATAAA7AACGCCATGCCGTCATCAGCGGTT CCGTCCTTTACAACCAAGACGAGAAAGGCAGTTA CCGCCTCGGCATCTTTGGCGGGCAAGCCCAGGAA GTTGCCGGCAGCGCGGAAGTGGAAACCGCAAACG GCATACACCTTATCGGTCTTGCCGCCAAGCAGTA AAGGCCTTCTAGAgacgATgccgtctgaagaaac cttgcgctcgttcgccagccaggacagaaatgcc t cgacttcgctgctgcccaaggttgccgggtgac gcacaccgtggaaacggatgaaggcacgaaccca gtggacataagcctgttcggttcgtaagctgtaa tgcaagtagcgtatgcgct cacgcaactggtcca gaaccttgaccgaacgcagcggtggtaacggcgc agtggcggttttcatggcttgttatgactgtttt tttggggtacagt ctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatAttorney Docket No. GMV0005-401-PCgttatggagcagcaacgatgttacgcagcagggc agtcgccctaaaacaaagttaaacatcatgaggg aagcggtgatcgccgaagtatcgactcaactatc agaggtagttggcgt cat cgagcgccatctcgaa ccgacgttgctggccgtacatttgtacggctccg cagtggatggcggcctgaagccacacagtgatat tgatttgctggttacggtgaccgtaaggcttgat gaaacaacgcggcgagctttgatcaacgaccttt tggaaacttcggcttcccctggagagagcgagat t ct ccgcgctgtagaagt caccattgttgtgcac gacgacatcattccgtggcgttatccagctaagc gcgaactgcaatttggagaatggcagcgcaatga cattcttgcaggtat ctt cgagccagccacgat c gacattgatctggctatcttgctgacaaaagcaa gagaacatagcgttgccttggtaggt ccagcggc ggaggaact ctttgat ccggtt cctgaacaggat ctatttgaggcgctaaatgaaaccttaacgctat ggaact cgccgcccgactgggctggcgatgagcg aaatgtagtgcttacgttgt cccgcatttggtac agcgcagtaaccggcaaaatcgcgccgaaggatg t cgctgccgactgggcaatggagcgcctgccggc ccagtatcagcccgt catacttgaagctagacag gcttatcttggacaagaagaagatcgcttggcct cgcgcgcagatcagttggaagaatttgtccacta cgtgaaaggcgagat caccaaggtagt cggcaaa taatgtctATgccgtctgaataCTCGAGcggaaC CCGGGTAGTGGCCTGTAAAAGACAGCATATAGAG AT GAGCAGGCT GT AT AAT AT T AAGGAT TT TT T T G T AACT T C T AT AAAT AT AAAGT AAT T T T T T AGGAG TTATATTATTAGGGCTTCTAGGAAGCTCAAATAG ATAAATAGATTCAAATAGATTCTTGTTAGCTGAT TGATGAACTAACTTAGGCATTTTTAAGTTTTTAG AAGTATATAAAATTACTAGTAAATTATTGGTTAA TTTTTGTATTTTAATTAGGCTTTGGACTTGGTTA AGCTGACCTAAATTAGATATGACAAATAAATTGT TACGTGGGGGGGTAAGATAAAATGGAGATGTTGT C AACC AC AT T G AAT CTT G AAAAAAC TTTTTAGGC TGAAAAAGAGCTTTTTTTATTTTCTTTAGCATTA TTGTATCTCTTAAAAATTAATGAGAATTAGCTAT ATGTAATAGCCAATCCTCTGTTAATAAAGTAACT AAGTTAATAAGCATTATTCAATATCAGTTTTTTT GATTTGAGCACCTTTGCGAATATTGCAAGCAGCG ACCTTACCAAATAATGTTTCATATTCGTTGACGC TGAAGTCTCCATTGCCTGGGCGTTTAACCCATAG GTTATCTAAGCTTGAGTATTCTATAGTGTCACCT AAATAGCTTGGCGTAATCATGGTCATAGCTGTTT CCTGTGTGAAATTGTTATCCGCTCACAATTCCAC ACAACATACGAGCCGGAAGCATAAAGTGTAAAGC CTGGGGTGCCTAATGAGTGAGCTAACTCACATTA ATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGG GAAACCTGTCGTGCCAGCTGCATTAATGAATCGG CCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGG CGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGC GCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAAAttorney Docket No. GMV0005-401-PCTCAGGGGATAACGCAGGAAAGAACATGTGAGCAA AAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGC CGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCC C C T GAG GAGCAT GAG AAAAAT C GAG GC T CAAG T C AGAGGTGGCGAAACCCGACAGGACTATAAAGATA CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC TCTCCTGTTCCGACCCTGCCGCTTACCGGATACC TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT TTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGC ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTT ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG TGCTACAGAGTTCTTGAAGTGGTGGCCTAACTAC GGCTACACTAGAAGAACAGTATTTGGTATCTGCG CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGT TGGTAGCTCTTGATCCGGCAAACAAACCACCGCT GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGA TTACGCGCAGAAAAAAAGGATCTCAAGAAGATCC TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG AACGAAAACTCACGTTAAGGGATTTTGGTCATGA GATTATCAAAAAGGATCTTCACCTAGATCCTTTT AAATTAAAAAT GAAGT TT TAAAT CAAT CTAAAGT ATATATGAGTAAACTTGGTCTGACAGTTACCAAT GCTTAATCAGTGAGGCACCTATCTCAGCGATCTG TCTATTTCGTTCATCCATAGTTGCCTGACTCCCC GTCGTGTAGATAACTACGATACGGGAGGGCTTAC CATCTGGCCCCAGTGCTGCAATGATACCGCGAGA CCCACGCTCACCGGCTCCAGATTTATCAGCAATA AACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTG GTCCTGCAACTTTATCCGCCTCCATCCAGTCTAT TAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCG CCAGTTAATAGTTTGCGCAACGTTGTTGCCATTG CTACAGGCATCGTGGTGTCACGCTCGTCGTTTGG TATGGCTTCATTCAGCTCCGGTTCCCAACGATCA AGGCGAGTTACATGATCCCCCATGTTGTGCAAAA AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGT CAGAAGTAAGTTGGCCGCAGTGTTATCACTCATG GTTATGGCAGCACTGCATAATTCTCTTACTGTCA TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA GTACTCAACCAAGTCATTCTGAGAATAGTGTATG CGGCGACCGAGTTGCTCTTGCCCGGCGTCAATAC GGGATAATACCGCGCCACATAGCAGAACTTTAAA AGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCA GTTCGATGTAACCCACTCGTGCACCCAACTGATC TTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAA AGGGAATAAGGGCGACACGGAAATGTTGAATACT CATACTCTTCCTTTTTCAATATTATTGAAGCATT TATCAGGGTTATTGTCTCATGAGCGGATACATAT T T GAAT GT ATT T AGAAAAAT AAACAAATAGGGGT TCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTAttorney Docket No. GMV0005-401-PCATAAAAATAGGCGTATCACGAGGCCCTTTCGTCT CGCGCGTTTCGGTGATGACGGTGAAAACCTCTGA CACATGCAGCTCCCGGAGACGGTCACAGCTTGTC TGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCA GGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGC TGGCTTAACTATGCGGCATCAGAGCAGATTGTAC TGAGAGTGCACCATATGCGGTGTGAAATACCGCA CAGATGCGTAAGGAGAAAATACCGCATCAGGAAA TTGTAAGCGTTAATATTTTGTTAAAATTCGCGTT AAAT T T T T GT T AAAT C AG C T C AT T T T T T AAC C AA TAGGCCGAAATCGGCAAAATCCCTTATAAATCAA AAGAATAGACCGAGATAGGGTTGAGTGTTGTTCC AGTTTGGAACAAGAGTCCACTATTAAAGAACGTG GACTCCAACGTCAAAGGGCGAAAAACCGTCTATC AGGGCGATGGCCCACTACGTGAACCATCACCCTA ATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCA CTAAATCGGAACCCTAAAGGGAGCCCCCGATTTA GAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAG AAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCT AGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCG TAACCACCACACCCGCCGCGCTTAATGCGCCGCT ACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCA ACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTC GCTATTACGCCAGCTGGCGAAAGGGGGATGTGCT GCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTT CCCAGTCACGACGTTGTAAAACGACGGCCAGTGA ATTGTAATACGACTCACTATACapsuleSEQ ID NO: 17 upstream ID9Forward ATACGCGGTCGAGTTCGGCGCapsuleupstream ID9SEQ ID NO: 18Reverse( 1200bp) CGGAATCCTCCGAGTCTTGTACTTG Capsuledownstream ID9SEQ ID NO: 19Forward(850bp) gat gagcgaaatgtagt gotta eg CapsuleSEQ ID NO: 20 downstream ID9Reverse GCATCTGTCGTAGCAGATAAAGACAttorney Docket No. GMV0005-401-PCt cgcgcgtttcggtgatgacggtgaaaacctctg acacatgcagctcccggagacggtcacagcttgt ctgtaagcggatgccgggagcagacaagcccgtc agggcgcgt cagcgggtgttggcgggtgt cgggg ctggcttaactatgcggcatcagagcagattgta ctgagagtgcaccatatgcggtgtgaaataccgc acagatgcgtaaggagaaaataccgcatcaggcg ccattcgccattcaggctgcgcaactgttgggaa gggcgatcggtgcgggcctcttcgctattacgcc agctggcgaaagggggatgtgctgcaaggcgatt aagttgggtaacgccagggttttcccagtcacga cgttgtaaaacgacggccagtgccaagcttATGT GTATCGAGATGAAATTTATATTTTTTGTACTGTA TGTTTTGCAGTTTCTGCCGTTTGCGCTGCTGCAC AAGATTGCCGACCTGACGGGTTTGCTTGCCTACC TTCTGGTCAAACCGCGCCGCCGTATCGGCGAAAT CAATTTGGCAAAATGTTTTTCCGAATGGAGTGAG GAAAAGCGTAAAACCGTGTTGAAACAGCATTTCA AACACATGGCGAAACTGATGTTGGAATACGGTTT ATATTGGTACGCGCCTGCCGGACGTTTGAAATCG CTGGTGCGCTACCGCAATAAGCATTATTTGGACG ACGCGCTGGCGGCGGGGGAAAAAGTCATCATCCT GTATCCGCACTTCACCGCTGCAGTTCTCGAGGCA GTGACTAACTAGGAGGAATAAATGGCTAAAATGA G AAT AT C AC CG GAAT T GAAAAAACT GAT C GAAAA ATACCGCTGCGTAAAAGATACGGAAGGAATGTCTpUC18 Plasmid CCTGCTAAGGTATATAAGCTGGTGGGAGAAAATG SEQ ID NO: 21 (IpxLl KO+FHbp AAAACCTATATTTAAAAATGACGGACAGCCGGTA ID9 SM) TAAAGGGACCACCTATGATGTGGAACGGGAAAAG GACATGATGCTATGGCTGGAAGGAAAGCTGCCTG TTCCAAAGGTCCTGCACTTTGAACGGCATGATGG CTGGAGCAATCTGCTCATGAGTGAGGCCGATGGC GTCCTTTGCTCGGAAGAGTATGAAGATGAACAAA GCCCTGAAAAGATTATCGAGCTGTATGCGGAGTG CATCAGGCTCTTTCACTCCATCGACATATCGGAT TGTCCCTATACGAATAGCTTAGACAGCCGCTTAG CCGAATTGGATTACTTACTGAATAACGATCTGGC CGATGTGGATTGCGAAAACTGGGAAGAAGACACT CCATTTAAAGATCCGCGCGAGCTGTATGATTTTT T AAAGAC G GAAAAGC C CG AAG AGGAAC T T GT C T T TTCCCACGGCGACCTGGGAGACAGCAACATCTTT GTGAAAGATGGCAAAGTAAGTGGCTTTATTGATC TTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGA CATTGCCTTCTGCGTCCGGTCGATCAGGGAGGAT ATCGGGGAAGAACAGTATGTCGAGCTATTTTTTG ACTTACTGGGGATCAAGCCTGATTGGGAGAAAAT AAAAT ATT ATAT T TT ACT GGAT GAAT T GT TT T AG TACCCCTCAGCGCATGCATCCGGGCAGCAGCGCA TCGGCTCGCACGAGGTCTGCGCTTGAATTGTGTT GT AGAAAC ACAAC GT T T T T GAAAAAAT AAGC T AT TGTTTTATAT C AAAAT AT AAT C AT T T T T AAAAT A AAGGTTGCGGCATTTATCAGATATTTGTTCTGAA ATGAAGACGTATCGGGTGTTTGCCCGATGTTTTT AGGTTTTTATCAAATTTACAAAAGGAAGCCCATATGGTGAACCGAACTGCCTTCTGCCTTTCTCTGACAttorney Docket No. GMV0005-401-PCCGCCGCCCTGATTCTGACCGCCTGCAGTAGCGGA GGCGGCGGAAGCGGAGGCGGCGGTGTCGCCGCCG ACATCGGTGCGGGGCTTGCTGATGCACTAACCGC ACCGCTCGACCATAAAGACAAAGGTTTGCAGTCT TTGACGCTGGATCAGTCCGTCAGGAAAAACGAGA AACTGAAGCTGGCGGCACAAGGTGCGGAAAAAAC TTATGGAAACGGCGACAGCCTCAATACGGGCAAA TTGAAGAACGACAAGGTCAGCCGCTTCGACTTTA TCCGTCAAATCGAAGTGGACGGGCAGCTCATTAC CTTGGAGAGCGGAGAGTTCCAAGTGTACAAACAA AGCCATTCCGCCTTAACCGCCCTTCAGACTGAGC AAGTACAAGACTCGGAGGATTCCGGGAAGATGGT TGCGAAACGCCAGTTCAGAATCGGCGACATAGCG GGCGAACATACGTCTTTTGACAAGCTTCCCAAAG GCGGCAGTGCGACATATCGCGGGACGGCGTTCGG T T C AG AC G AT G C T GG C GG AAAAC T G AC C T AT AC T ATAGATTTCGCCGTCAAACAGGGACACGGCAAAA T C GAAC AT T T GAAAT C GC C C GAACT C AAT GT C GA CCTGGCCGCCGCCTATATCAAGCCGGATAAAAAA CGCCATGCCGTCATCAGCGGTTCCGTCCTTTACA ACCAAGACGAGAAAGGCAGTTACCGCCTCGGCAT CTTTGGCGGGCAAGCCCAGGAAGTTGCCGGCAGC GCGGAAGTGGAAACCGCAAACGGCATACACCTTA TCGGTCTTGCCGCCAAGCAGTAAAGGCCTCCTGC AGGCCTGCAGGGAATAATGAGTCGACAGGATTTC GGACGCAACGATTCGGTTTTTGTGGATTTTTTCG GTATTCAGACGGCAACGATTACCGGATTGAGCCG CATTGCCGCGCTTGCAAATGCAAAAGTGATACCC GCCATTCCCGTCCGCGAGGCAGACAATACGGTTA CATTGCATTTCTACCCTGCTTGGAAATCCTTTCC GGGTGAAGACGCGAAAGCCGACGCGCAGCGCATG AACCGTTTTATCGAAGACAGGGTGCGCGAACATC CGGAACAATATTTTTGGCTGCACAAGCGTTTTAA AACCCGTCCGGAAGGCAGCCCCGATTTTTACTGA CTACGTCAGACGGCtctagaggatccccgggtac cgagct cgaatt cgtaat catggtcatagctgtt t cctgtgtgaaattgttat ccgctcacaattcca cacaacatacgagccggaagcataaagtgtaaag cctggggtgcctaatgagtgagctaactcacatt aattgcgttgcgctcactgcccgcttt ccagt eg ggaaacctgtcgtgccagctgcattaatgaatcg gccaacgcgcggggagaggcggtttgcgtattgg geget ett ccgcttcctcgctcactgact egetg cgctcggtcgttcggctgcggcgagcggtatcag ct cact caaaggcggtaatacggttat ccacaga at caggggataacgcaggaaagaacatgtgagca aaaggccagcaaaaggccaggaaccgtaaaaagg ccgcgttgctggcgttttt ccataggctccgccc ccctgacgagcat cacaaaaat egaeget caagt cagaggtggcgaaacccgacaggactataaagat accaggcgtttccccctggaagctccctcgtgcg ctctcctgttccgaccctgccgcttaccggatac ctgtccgcctttctcccttcgggaagcgtggcgc tttctcaaagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgAttorney Docket No. GMV0005-401-PCcacgaaccccccgttcagcccgaccgctgcgcct tatccggtaactatcgtcttgagtccaacccggt aagacacgacttatcgccactggcagcagccact ggtaacaggattagcagagcgaggtatgtaggcg gtgctacagagttcttgaagtggtggcctaacta cggctacactagaagaacagtatttggtatctgc got ctgctgaagccagttacctt cggaaaaagag ttggtagctcttgatccggcaaacaaaccaccgc tggtagcggtggtttttttgtttgcaagcagcag attacgcgcagaaaaaaaggat ctcaagaagat c ctttgatcttttctacggggtctgacgctcagtg gaacgaaaactcacgttaagggattttggtcatg agattatcaaaaaggatctt cacctagat ccttt taaattaaaaatgaagttttaaatcaatctaaag tatatatgagtaaacttggt ctgacagttaccaa tgcttaat cagtgaggcacctat ct cagcgat ct gtctatttcgttcatccatagttgcctgactccc cgt cgtgtagataactacgatacgggagggctta ccatctggccccagtgctgcaatgataccgcgag acccacgctcaccggctccagatttatcagcaat aaac cage cage cggaagggccgagcgcagaagt ggt cctgcaactttat ccgcct ccat ccagtcta ttaattgttgccgggaagctagagtaagtagttc gccagttaatagtttgcgcaacgttgttgccatt gctacaggcatcgtggtgt cacgct cgtcgtttg gtatggcttcattcagctccggttcccaacgatc aaggcgagttacatgatcccccatgttgtgcaaa aaagcggttagct ccttcggtcctccgat cgttg tcagaagtaagttggccgcagtgttatcactcat ggttatggcagcactgcataatt ct ettaetgt c atgccatccgtaagatgctttt ctgtgactggtg agtactcaaccaagtcattctgagaatagtgtat gcggcgaccgagttgctcttgcccggcgt caata cgggataataccgcgccacatagcagaactttaa aagtgctcatcattggaaaacgttcttcggggcg aaaactctcaaggatcttaccgctgtt gagatec agttcgatgtaacccact cgtgcacccaactgat cttcagcatcttttactttcaccagcgtttctgg gtgagcaaaaacaggaaggcaaaatgccgcaaaa aagggaataagggcgacacggaaatgttgaatac tcatactcttcctttttcaatattattgaagcat ttatcagggttattgt ct catgagcggatacata tttgaatgtatttagaaaaataaacaaatagggg ttccgcgcacatttccccgaaaagtgccacctga cgt ctaagaaaccattattatcatgacattaacc tataaaaataggcgtatcacgaggccctttcgt c Lpxll upstreamSEQ ID NO: 22ID9 Forward GTCGGCAAGGGTAAGGCAGCLpxll upstreamSEQ ID NO: 23ID9 Reverse CCCTTTATACCGGCTGTCCGLpxlldownstream ID9SEQ ID NO: 24Forward( 68 Obp ) TTCCGTCCTTTACAACCAAAttorney Docket No. GMV0005-401-PCLpxllSEQ ID NO: 25 downstream ID9Reverse atattcataggtttgcggtatttc CCTCAGCGCATGCATCCGGGCAGCAGCGCATCGG CTCGCACGAGGTCTGCGCTTGAATTGTGTTGTAG AAACACAACGT T T TT GAAAAAAT AAGCTATT GT T T TAT AT C AAAAT AT AAT 0 AT T T T T AAAAT AAAGG TTGCGGCATTTATCAGATATTTGTTCTGAAATGA AGACGTATCGGGTGTTTGCCCGATGTTTTTAGGT T T T T AT C AAAT T T AC AAAAGG AAGC C CAT AT G GT GAACCGAACTGCCTTCTGCCTTTCTCTGACCGCC GCCCTGATTCTGACCGCCTGCAGTAGCGGAGGCG GCGGAAGCGGAGGCGGCGGTGTCGCCGCCGACAT CGGTGCGGGGCTTGCTGATGCACTAACCGCACCG CTCGACCATAAAGACAAAGGTTTGCAGTCTTTGA C GC T GGAT C AG T C C G T CAGGAAAAAC GAGAAAC T GAAGCTGGCGGCACAAGGTGCGGAAAAAACTTAT GGAAACGGCGACAGCCTCAATACGGGCAAATTGA AGAACGACAAGGTCAGCCGCTTCGACTTTATCCG TCAAATCGAAGTGGACGGGCAGCTCATTACCTTG GAGAGCGGAGAGTTCCAAGTGTACAAACAAAGCC ATTCCGCCTTAACCGCCCTTCAGACTGAGCAAGT ACAAGACTCGGAGGATTCCGGGAAGATGGTTGCG AAACGCCAGTTCAGAATCGGCGACATAGCGGGCG AACATACGTCTTTTGACAAGCTTCCCAAAGGCGG CAGTGCGACATATCGCGGGACGGCGTTCGGTTCA GACGATGCTGGCGGAAAACTGACCTATACTATAGBlue script ATTTCGCCGTCAAACAGGGACACGGCAAAATCGA Plasmid (FHbp ACATTTGAAATCGCCCGAACTCAATGTCGACCTG SEQ ID NO: 26KO+FHbp ID22 GCCGCCGCCTATATCAAGCCGGATAAAAAACGCC TM) ATGCCGTCATCAGCGGTTCCGTCCTTTACAACCA AGACGAGAAAGGCAGTTACCGCCTCGGCATCTTT GGCGGGCAAGCCCAGGAAGTTGCCGGCAGCGCGG AAGTGGAAACCGCAAACGGCATACACCTTATCGG TCTTGCCGCCAAGCAGTAAAGGCCTCCTGCAGGC C AAGCAAAACC AT TGT GAAAAT GCC GT CC GAACA CGATAATTTACCGTTCGGACGGCATTTTGTATTG CACCGTCCGACGGCATGCCCAAGGGGGGAAATCC CTATTTTCAGGCCAACCGCTATATAATGCCGTCT GAACC AAC GAG AGAAT GC C AT GCAAGC T GAT T T T AACCGTCCCGTCCTGGCCGTCGATACCGGTACTT CCCGTTTGTCGCTCGCGCTGCGTGCCGACGGCGA AACCCGTCTGTTCCATCAGGAAGTCGGCAGCCGC CAGTCCGAACTGATTCTGCCGGAAATCCGCACCC TATTCCGCGATGCAGGCATTACCGCCGCCGATTT GGGTGCGGTCGTGTACGCACAGGGTCCCGGCGCG TTTACCGGACTGCGTATCGGCATCGGTGTAGCTC AGGGTTTGGCAACGCCGTTTGATACCCCCTTAAT CGGCGTACCCTCGCTCGATGCCGCCGCCTCGCTG CCGCCGCCGCAAAGCTGCATCCTTGCCGCTACGG ACGCTCGTATGGGCGAAGTGTTTTATGCATGGTT CGATACGCTGCTCGAGGGGGGGCCCGGTACCAGC TTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCT TGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAAT T GT T AT C C GCT C AC AAT T C CAC AC AAC AT AAttorney Docket No. GMV0005-401-PCCGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTG CCTAATGAGTGAGCTAACTCACATTAATTGCGTT GCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTG TCGTGCCAGCTGCATTAATGAATCGGCCAACGCG CGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTC GTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA AGGCGGTAATACGGTTATCCACAGAATCAGGGGA TAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG C AAAAGGC C AG G AAC C GT AAAAAGG C C GC GT T GC TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA GCATCACAAAAATCGACGCTCAAGTCAGAGGTGG CGAAACCCGACAGGACTATAAAGATACCAGGCGT TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGT TCCGACCCTGCCGCTTACCGGATACCTGTCCGCC TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATA GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC CCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGA CTTATCGCCACTGGCAGCAGCCACTGGTAACAGG ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG AGTTCTTGAAGTGGTGGCCTAACTACGGCTACAC TAGAAGAACAGTATTTGGTATCTGCGCTCTGCTG AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT CTTGATCCGGCAAACAAACCACCGCTGGTAGCGG TGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGC AGAAAAAAAGG AT C T CAAGAAG AT C C T T T GAT C T TTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCA AAAAGGAT C T T C AC C T AG AT C C T T T T AAAT T AAA AAT GAAGT T TT AAAT CAAT CTAAAGT AT ATAT GA GTAAACTTGGTCTGACAGTTACCAATGCTTAATC AGTGAGGCACCTATCTCAGCGATCTGTCTATTTC GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA GATAACTACGATACGGGAGGGCTTACCATCTGGC CCCAGTGCTGCAATGATACCGCGAGACCCACGCT CACCGGCTCCAGATTTATCAGCAATAAACCAGCC AGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCA ACTTTATCCGCCTCCATCCAGTCTATTAATTGTT GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAA TAGTTTGCGCAACGTTGTTGCCATTGCTACAGGC ATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTT CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGT TACATGATCCCCCATGTTGTGCAAAAAAGCGGTT AGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA AGTTGGCCGCAGTGTTATCACTCATGGTTATGGC AGCACTGCATAATTCTCTTACTGTCATGCCATCC GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACC GAGTTGCTCTTGCCCGGCGTCAATACGGGATAAT ACCGCGCCACATAGCAGAACTTTAAAAGTGCTCA TCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC AAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATAttorney Docket No. GMV0005-401-PCCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATA AGGGCGACACGGAAATGTTGAATACTCATACTCT TCCTTTTTCAATATTATTGAAGCATTTATCAGGG TTATTGTCTCATGAGCGGATACATATTTGAATGT ATTTAGAAAAATAAACAAATAGGGGTTCCGCGCA C AT T T C C C C GAAAAG T GC C AC C T AAAT T G T AAGC GT T AAT AT T T T GT T AAAAT T CGC GT T AAAT T T T T GTTAAATCAGCTCATTTTTTAACCAATAGGCCGA AAT C G GC AAAAT C C C T TAT AAAT CAAAAGAAT AG ACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGA ACAAGAGTCCACTATTAAAGAACGTGGACTCCAA C GT C AAAG GGC G AAAAAC C GT C T AT C AGG GC G AT GGCCCACTACGTGAACCATCACCCTAATCAAGTT TTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCG GAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGA CGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAG GGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCT GGCAAGTGTAGCGGTCACGCTGCGCGTAACCACC ACACCCGCCGCGCTTAATGCGCCGCTACAGGGCG CGTCCCATTCGCCATTCAGGCTGCGCAACTGTTG GGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTA CGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTC ACGACGTTGTAAAACGACGGCCAGTGAATTGTAA TACGACTCACTATAGGGCGAATTGGAGCTCCACC GCGGTGGCGGCCGCTCTAGACCAGCCACGGCGCA TACAAATTCACCCGTCCGCCCACAGGCGATGTAT TACGTATCGACCGCATCAAAGAAATCCACCAAGC CCTGCCCAATACACACATCGTGATGCACGGCTCC AGCTCCGTTCCGCAAGAATGGCTGAAAGTCATCA ACGAATACGGCGGCAATATCGGCGAAACCTACGG CGTGCCGGTTGAAGAAATCGTCGAAGGCATCAAA CACGGCGTGCGCAAAGTCAACATCGATACCGACT TGCGCCTTGCTTCTACCGGCGCGGTACGCCGCTA CCTTGCCGAAAATCCGTCCGACTTTGACCCGCGC AAAT AC C T GAG C AAAACC AT T GAGG C C AT GAAGC AAATCTGCCTCGACCGTTATCTTGCGTTTGGCTG CGAAGGTCAGGCAGGCAAAATCAAACCTGTTTCG TTGGAAAAAATGGCAAGCCGTTATGCCAAGGGCG AAT T GAAC CAAAT C GT CAAAT AACAGGT T GC C T G TAAACAAAATGCCGTCCCATGGGCAAACTTAAGA GTGTGTTGATAGTGCAGTATCTTAAAATTTTGTG TATAATAGGAATTGAAGTTAAATTAGATGCTAAA AATTTGTAATTAAGAAGGAGGGATTCGTCATGTT GGTATTCCAAATGCGTAATGTAGATAAAACATCT ACTGTTTTGAAACAGACTAAAAACAGTGATTACG C AGATAAAT AAAT AC GTT AGAT T AAT T CC TAG CA GTGACTAATCTTATGACTTTTTAAACAGATAACT AAAAT TACAAA CAAAT CGT T TAACT T CTGTAT T T ATTTACAGATGTAATCACTTCAGGAGTAATTACA T GAAC AAAAAT AT AAAAT AT T C T CAAAAC T T T T T AACGAGTGAAAAAGTACTCAACCAAATAATAAAA CAATTGAATTTAAAAGAAACCGATACCGTTTACGAAATTGGAACAGGTAAAGGGCATTTAACGACGAAAttorney Docket No. GMV0005-401-PCACTGGCTAAAATAAGTAAACAGGTAACGTCTATT GAAT T AGAC AG T C AT C T AT T C AACT T AT C GT C AG AAAAAT T AAAAC T GAACAT T C GT GT C AC T T T AAT TCACCAAGATATTCTACAGTTTCAATTCCCTAAC AAACAGAGGTATAAAATTGTTGGGAGTATTCCTT ACCATTTAAGCACACAAATTATTAAAAAAGTGGT TTTTGAAAGCCATGCGTCTGACATCTATCTGATT GTTGAAGAAGGATTCTACAAGCGTACCTTGGATA TTCACCGAACACTAGGGTTGCTCTTGCACACTCA AGTCTCGATTCAGCAATTGCTTAAGCTGCCAGCG GAATGCTTTCATCCTAAACCAAAAGTAAACAGTG TCTTAATAAAACTTACCCGCCATACCACAGATGT T C C AG AT AAAT AT T G GAAG C T AT AT AC GT AC T T T GTTTCAAAATGGGTCAATCGAGAATATCGTCAAC T GT T T AC T AAAAAT C AGT T T C AT CAAGC AAT GAA ACACGCCAAAGTAAACAATTTAAGTACCATTACT TATGAGCAAGTATTGTCTATTTTTAATAGTTATC TATTATTTAACGGGAGGAAATAATTCTATGAGTC GCTTTTTTAAATTTGGAAAGTTACACGTTACTAA AGGGAATGGAGATAAATTATTAGATATACTACTG ACAGCTTCCAAGAAGCTAAAGAGGTCCCTTTCGA CGGCCCCGGGFHbp ID22SEQ ID NO: 27 DownstreamForward GAAATGAAGACGTATCGGGTGFHbpID22SEQ ID NO: 28 DownstreamReverse TTCGTGAACCTTTTCCCTTATCFHbp ID22SEQ ID NO: 29 UpstreamForward GGCGAAATCGGCGTATTGGGFHbp ID22SEQ ID NO: 30 UpstreamReverse CTACATTACGCATT T GGAAT AC CAttorney Docket No. GMV0005-401-PCGGGCGAATTCCTGGACACGGCAGACCGATAAAGC CCGCACCGCCGGTAACGAGGATGGTTTTTTTGTT TGCGGTTTGCATTTAATCCCCATATCCGTTGGGG TTGCGGCTGACCCAACGCCACGAATCTTCCATCA TTTGCTGCAAGCCGCGTTTGGTTTCCCAGCCGGT TTGTTGTTTGGTATGGGACGGGTCGGCAAAGGAA CACGCCAAATCACCGGCGCGGCGCGGTTTGATTT CAAATGGAATGGTTAAACCCGAAGCCGCTTCAAA TGCGCGGATGATTTCCAATACCGAAGAAGCGCGG CCGGAGCCTAAGTTCAGCAAATGCGTGCCTGCTA CATTACTTTTTGCCTGCATAGCCGCGACATGGCC TTCTGCCAAATCCATCACATGAATATAGTCACGC ATCCCCGTGCCGTCGGGGGTAGGGTAGTCATCGC CAAATACCGCCAATTGCCTCAGCGCATGCATCCG GGCTCTGCGCTTGAATTGTGTTGTAGAAACACAA C GT TT T T G AAAAAAT AAG CTATTGTTTTATATCA AAATAT AAT CAT T TT TAAAAT AAAGGT TGCGGCA TTTATCAGATATTTGTTCTGAAATGAAGACGTAT CGGGTGTTTGCCCGATGTTTTTAGGTTTTTATCA AATTTACAAAAGGAAGCCCATATGCGGTATGTAA AAAAAGATACAATAACTAGGGTGTTTATATATTA TCTATATCTGCGTATGACTAGGAGCAAACCTGTG AACCGAACTACCTTCTGTTGCCTTTCTCTGACCG CCGCCCTGATTCTGACCGCCTGCAGCAGCGGAGG GGGCGGTGTCGCCGCCGACATCGGCGCGGGGCTT GCCGATGCACTAACCGCACCGCTCGACCATAAAGpGEM Plasmid ACAAAAGTTTGCAGTCTTTGACGCTGGATCAGTC(CapsuleSEQ ID NO: 31 CGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCA KO+FHbp ID22CAAGGTGCGGAAAAAACTTATGGAAACGGCGACA TM)GCCTCAATACGGGCAAATTGAAGAACGACAAGGT CAGCCGCTTCGACTTTATCCGTCAAATCGAAGTG GACGGGCAGCTCATTACCTTGGAGAGCGGAGAGT TCCAAATATACAAACAGGACCACTCCGCCGTCGT T GC CC T AC AGAT T GAAAAAAT C AAC AAC C CC G AC AAAAT C G AC AGC C T GAT AAAC CAAC GC T C CT T C C GTGTCAGCGATTTGGGTGGAGAACATACCGCCTT CAACCAACTGCCCAGCGGCAAAGCCGAGTATCAC GGCAAAGCATTCAGCTCCGACGACCCGAACGGCA GGCTGCACTACTCCATTGATTTTACCAAAAAACA GGGTTACGGCAGAATCGAACACCTGAAAACGCCC GAGCAGAATGTCGAGCTTGCCTCCGCCGAACTCA AAGCAGATGAAAAATCACACGCCGTCATTTTGGG CGACACGCGCTACGGCGGCGAAGAAAACGGCACT TACCACCTCGCCCTTTTCGGCGACCGCGCCCAAG AAATCGCCGGCTCGGCAACCGTGAAGATAAGGGA AAAGGTTCACGAAATCGGCATCGCCGGCAAACAG TAGAGGCCTTCTAGAgacgATgccgt ctgaagaa accttgcgctcgttcgccagccaggacagaaatg cct cgactt cgctgctgcccaaggttgccgggtg acgcacaccgtggaaacggatgaaggcacgaacc cagtggacataagcctgtt cggttcgtaagctgt aatgcaagtagcgtatgcgctcacgcaactggtc cagaaccttgaccgaacgcagcggtggtaacggc gcagtggcggttttcatggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaaAttorney Docket No. GMV0005-401-PCgcagcaagcgcgttacgccgtgggt cgatgtttg atgttatggagcagcaacgatgttacgcagcagg gcagtcgccctaaaacaaagttaaacatcatgag ggaagcggtgat cgccgaagtat cgactcaacta tcagaggtagttggcgtcatcgagcgccatctcg aaccgacgttgctggccgtacatttgtacggctc cgcagtggatggcggcctgaagccacacagtgat attgatttgctggttacggtgaccgtaaggcttg atgaaacaacgcggcgagctttgatcaacgacct tttggaaacttcggcttcccctggagagagcgag attctccgcgctgtagaagtcaccattgttgtgc acgacgacatcattccgtggcgttatccagctaa gcgcgaactgcaatttggagaatggcagcgcaat gacattcttgcaggtatcttcgagccagccacga t cgacattgatctggctat cttgctgacaaaagc aagagaacatagcgttgccttggtaggtccagcg gcggaggaactctttgatccggttcctgaacagg at ctatttgaggcgctaaatgaaaccttaacgct atggaact cgccgcccgactgggctggcgatgag cgaaatgtagtgcttacgttgtcccgcatttggt acagcgcagtaaccggcaaaat cgcgccgaagga tgt cgctgccgactgggcaatggagcgcctgccg gcccagtatcagcccgtcatacttgaagctagac aggcttat cttggacaagaagaagat cgcttggc ct cgcgcgcagat cagttggaagaatttgtccac tacgtgaaaggcgagatcaccaaggtagtcggca aataatgt ctATgccgtctgaataCTCGAGcgga aCCCGGGTAGTGGCCTGTAAAAGACAGCATATAG AGATGAGCAGGCTGTATAATATTAAGGATTTTTT TGTAACTTCTATAAATATAAAGTAATTTTTTAGG AGT T AT AT T AT T AGGGCT T CT AGGAAGCT CAAAT AGAT AAAT AGAT T C AAAT AGAT T CT T GT T AGC T G ATTGATGAACTAACTTAGGCATTTTTAAGTTTTT AGAAGT AT AT AAAAT T AC T AGT AAAT TATTGGTT AATTTTTGTATTTTAATTAGGCTTTGGACTTGGT TAAGCTGACCTAAATTAGATATGACAAATAAATT GTTACGTGGGGGGGTAAGATAAAATGGAGATGTT GT C AAC C AC AT T GAAT CT T GAAAAAAC T T T T T AG GCTGAAAAAGAGCTTTTTTTATTTTCTTTAGCAT TATTGTATCTCTTAAAAATTAATGAGAATTAGCT ATATGTAATAGCCAATCCTCTGTTAATAAAGTAA C T AAGT TAATAAGCAT TAT T CAATAT CAGTT T T T TTGATTTGAGCACCTTTGCGAATATTGCAAGCAG CGACCTTACCAAATAATGTTTCATATTCGTTGAC GCTGAAGTCTCCATTGCCTGGGCGTTTAACCCAT AGGTTATCTAAGCTTGAGTATTCTATAGTGTCAC CTAAATAGCTTGGCGTAATCATGGTCATAGCTGT TTCCTGTGTGAAATTGTTATCCGCTCACAATTCC ACACAACATACGAGCCGGAAGCATAAAGTGTAAA GCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTC GGGAAACCTGTCGTGCCAGCTGCATTAATGAATC GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTG GGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAAttorney Docket No. GMV0005-401-PCGCTCACTCAAAGGCGGTAATACGGTTATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGC AAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAG GCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC CCCCTGACGAGCATCACAAAAATCGACGCTCAAG TCAGAGGTGGCGAAACCCGACAGGACTATAAAGA TACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGC GCTCTCCTGTTCCGACCCTGCCGCTTACCGGATA CCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG CTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGT GCACGAACCCCCCGTTCAGCCCGACCGCTGCGCC TTATCCGGTAACTATCGTCTTGAGTCCAACCCGG TAAGACACGACTTATCGCCACTGGCAGCAGCCAC TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT ACGGCTACACTAGAAGAACAGTATTTGGTATCTG CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGA GTTGGTAGCTCTTGATCCGGCAAACAAACCACCG CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCA GATTACGCGCAGAAAAAAAGGATCTCAAGAAGAT CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT GGAACGAAAACTCACGTTAAGGGATTTTGGTCAT GAGATTATCAAAAAGGATCTTCACCTAGATCCTT T T AAAT T AAAAAT G AAGT T T T AAAT C AAT CT AAA GTATATATGAGTAAACTTGGTCTGACAGTTACCA ATGCTTAATCAGTGAGGCACCTATCTCAGCGATC TGTCTATTTCGTTCATCCATAGTTGCCTGACTCC CCGTCGTGTAGATAACTACGATACGGGAGGGCTT ACCATCTGGCCCCAGTGCTGCAATGATACCGCGA GACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT TGCTACAGGCATCGTGGTGTCACGCTCGTCGTTT GGTATGGCTTCATTCAGCTCCGGTTCCCAACGAT CAAGGCGAGTTACATGATCCCCCATGTTGTGCAA AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCA TGGTTATGGCAGCACTGCATAATTCTCTTACTGT CATGCCATCCGTAAGATGCTTTTCTGTGACTGGT GAGT AC T C AAC C AAGT CAT T C T GAGAAT AGT GT A TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAAT ACGGGATAATACCGCGCCACATAGCAGAACTTTA AAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC GAAAACTCTCAAGGATCTTACCGCTGTTGAGATC CAGTTCGATGTAACCCACTCGTGCACCCAACTGA TCTTCAGCATCTTTTACTTTCACCAGCGTTTCTG GGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAA AAAGGGAATAAGGGCGACACGGAAATGTTGAATA CTCATACTCTTCCTTTTTCAATATTATTGAAGCA TTTATCAGGGTTATTGTCTCATGAGCGGATACAT AT T TGAAT GTAT T T AGAAAAAT AAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAttorney Docket No. GMV0005-401-PCACGTCTAAGAAACCATTATTATCATGACATTAAC CTATAAAAATAGGCGTATCACGAGGCCCTTTCGT CTCGCGCGTTTCGGTGATGACGGTGAAAACCTCT GACACATGCAGCTCCCGGAGACGGTCACAGCTTG TCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGT CAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGG GCTGGCTTAACTATGCGGCATCAGAGCAGATTGT ACTGAGAGTGCACCATATGCGGTGTGAAATACCG CACAGATGCGTAAGGAGAAAATACCGCATCAGGA AATTGTAAGCGTTAATATTTTGTTAAAATTCGCG TTAAATTTTTGTTAAATCAGCTCATTTTTTAACC AATAGGCCGAAATCGGCAAAATCCCTTATAAATC AAAAGAATAGACCGAGATAGGGTTGAGTGTTGTT CCAGTTTGGAACAAGAGTCCACTATTAAAGAACG TGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA TCAGGGCGATGGCCCACTACGTGAACCATCACCC TAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAG CACTAAATCGGAACCCTAAAGGGAGCCCCCGATT TAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCG AGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCG CTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCG CGTAACCACCACACCCGCCGCGCTTAATGCGCCG CTACAGGGCGCGTCCATTCGCCATTCAGGCTGCG CAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCT TCGCTATTACGCCAGCTGGCGAAAGGGGGATGTG CTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTT TTCCCAGTCACGACGTTGTAAAACGACGGCCAGT G AAT T GT AAT AC GAC T CAC T AT ACapsuleSEQ ID NO: 32 upstream ID22Forward ATACGCGGTCGAGTTCGGCGCapsuleSEQ ID NO: 33 upstream ID22Reverse AGGTTTGCTCCTAGTCATACGCAG CapsuleSEQ ID NO: 34 downstreamID22 Forward gat gagcgaaatgtagt gotta eg CapsuleSEQ ID NO: 35 downstreamID22 Reverse GCATCTGTCGTAGCAGATAAAGAC tcgcgcgtttcggtgatgacggtgaaaacctctg acacatgcagctcccggagacggtcacagcttgt ctgtaagcggatgccgggagcagacaagcccgt c agggcgcgtcagcgggtgttggcgggtgtcgggg pUC18 Plasmidctggcttaactatgcggcat cagagcagattgta SEQ ID NO: 36 (IpxLl KO+FHbp ctgagagtgcaccatatgcggtgtgaaataccgc ID22 TM)acagatgcgtaaggagaaaataccgcatcaggcg ccatt cgccatt caggctgcgcaactgttgggaa gggcgatcggtgcgggcct ett cgctattacgccagctggcgaaagggggatgtgctgcaaggcgattAttorney Docket No. GMV0005-401-PCaagttgggtaacgccagggtttt cccagt cacga cgttgtaaaacgacggccagtgccaagcttATGT GTATCGAGATGAAATTTATATTTTTTGTACTGTA TGTTTTGCAGTTTCTGCCGTTTGCGCTGCTGCAC AAGATTGCCGACCTGACGGGTTTGCTTGCCTACC TTCTGGTCAAACCGCGCCGCCGTATCGGCGAAAT CAATTTGGCAAAATGTTTTTCCGAATGGAGTGAG GAAAAGCGTAAAACCGTGTTGAAACAGCATTTCA AACACATGGCGAAACTGATGTTGGAATACGGTTT ATATTGGTACGCGCCTGCCGGACGTTTGAAATCG CTGGTGCGCTACCGCAATAAGCATTATTTGGACG ACGCGCTGGCGGCGGGGGAAAAAGTCATCATCCT GTATCCGCACTTCACCGCTGCAGTTCTCGAGGCA GTGACTAACTAGGAGGAATAAATGGCTAAAATGA GAATAT CACCGGAAT T GAAAAAACT GATCGAAAA ATACCGCTGCGTAAAAGATACGGAAGGAATGTCT CCTGCTAAGGTATATAAGCTGGTGGGAGAAAATG AAAACCTATATTTAAAAATGACGGACAGCCGGTA TAAAGGGACCACCTATGATGTGGAACGGGAAAAG GACATGATGCTATGGCTGGAAGGAAAGCTGCCTG TTCCAAAGGTCCTGCACTTTGAACGGCATGATGG CTGGAGCAATCTGCTCATGAGTGAGGCCGATGGC GTCCTTTGCTCGGAAGAGTATGAAGATGAACAAA GCCCTGAAAAGATTATCGAGCTGTATGCGGAGTG CATCAGGCTCTTTCACTCCATCGACATATCGGAT TGTCCCTATACGAATAGCTTAGACAGCCGCTTAG CCGAATTGGATTACTTACTGAATAACGATCTGGC CGATGTGGATTGCGAAAACTGGGAAGAAGACACT CCATTTAAAGATCCGCGCGAGCTGTATGATTTTT TAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTT TTCCCACGGCGACCTGGGAGACAGCAACATCTTT GTGAAAGATGGCAAAGTAAGTGGCTTTATTGATC TTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGA CATTGCCTTCTGCGTCCGGTCGATCAGGGAGGAT ATCGGGGAAGAACAGTATGTCGAGCTATTTTTTG ACTTACTGGGGATCAAGCCTGATTGGGAGAAAAT AAAAT AT TATATTTTACTGGAT GAAT TGTTTTAG TACCCCTCAGCGCATGCATCCGGGCAGCAGCGCA TCGGCTCGCACGAGGTCTGCGCTTGAATTGTGTT G T AG AAAC ACAAC GT T T T T GAAAAAAT AAGC T AT T GT T T T AT AT C AAAAT AT AAT C AT T T T T AAAAT A AAGGTTGCGGCATTTATCAGATATTTGTTCTGAA ATGAAGACGTATCGGGTGTTTGCCCGATGTTTTT AGGTTTTTATCAAATTTACAAAAGGAAGCCCATA TGGTGAACCGAACTGCCTTCTGCCTTTCTCTGAC CGCCGCCCTGATTCTGACCGCCTGCAGTAGCGGA GGCGGCGGAAGCGGAGGCGGCGGTGTCGCCGCCG ACATCGGTGCGGGGCTTGCTGATGCACTAACCGC ACCGCTCGACCATAAAGACAAAGGTTTGCAGTCT TTGACGCTGGATCAGTCCGTCAGGAAAAACGAGA AAC T GAAG C T G GC GG C AC AAGG T GC GGAAAAAAC TTATGGAAACGGCGACAGCCTCAATACGGGCAAA TTGAAGAACGACAAGGTCAGCCGCTTCGACTTTA TCCGTCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGCGGAGAGTTCCAAGTGTACAAACAAAttorney Docket No. GMV0005-401-PCAGCCATTCCGCCTTAACCGCCCTTCAGACTGAGC AAGTACAAGACTCGGAGGATTCCGGGAAGATGGT TGCGAAACGCCAGTTCAGAATCGGCGACATAGCG GGCGAACATACGTCTTTTGACAAGCTTCCCAAAG GCGGCAGTGCGACATATCGCGGGACGGCGTTCGG TTCAGACGATGCTGGCGGAAAACTGACCTATACT ATAGATTTCGCCGTCAAACAGGGACACGGCAAAA TCGAACATTTGAAATCGCCCGAACTCAATGTCGA CCTGGCCGCCGCCTATATCAAGCCGGATAAAAAA CGCCATGCCGTCATCAGCGGTTCCGTCCTTTACA ACCAAGACGAGAAAGGCAGTTACCGCCTCGGCAT CTTTGGCGGGCAAGCCCAGGAAGTTGCCGGCAGC GCGGAAGTGGAAACCGCAAACGGCATACACCTTA TCGGTCTTGCCGCCAAGCAGTAAAGGCCTCCTGC AGGCCTGCAGGGAATAATGAGTCGACAGGATTTC GGACGCAACGATTCGGTTTTTGTGGATTTTTTCG GTATTCAGACGGCAACGATTACCGGATTGAGCCG CATTGCCGCGCTTGCAAATGCAAAAGTGATACCC GCCATTCCCGTCCGCGAGGCAGACAATACGGTTA CATTGCATTTCTACCCTGCTTGGAAATCCTTTCC GGGTGAAGACGCGAAAGCCGACGCGCAGCGCATG AACCGTTTTATCGAAGACAGGGTGCGCGAACATC CGGAACAATATTTTTGGCTGCACAAGCGTTTTAA AACCCGTCCGGAAGGCAGCCCCGATTTTTACTGA CTACGTCAGACGGCt ctagaggatccccgggtac cgagctcgaattcgtaatcatggtcatagctgtt t cctgtgtgaaattgttat ccgctcacaattcca cacaacatacgagccggaagcataaagtgtaaag cctggggtgcctaatgagtgagctaactcacatt aattgcgttgcgctcactgcccgcttt ccagt eg ggaaacctgtcgtgccagctgcattaatgaat eg gccaacgcgcggggagaggcggtttgcgtattgg geget cttccgctt cct eget cactgact eget g egeteggt cgtt cggctgcggcgagcggtatcag ctcactcaaaggcggtaatacggttatccacaga at caggggataacgcaggaaagaacatgtgagca aaaggccagcaaaaggccaggaaccgtaaaaagg ccgcgttgctggcgtttttccataggctccgccc c c ct ga egage at cacaaaaatcga eget caagt cagaggtggcgaaacccgacaggactataaagat accaggcgtttccccctggaagctccctcgtgcg ct ctcctgttccgaccctgccgcttaccggatac ctgtccgccttt ctccctt cgggaagcgtggcgc tttctcaaagctcacgctgtaggtatctcagttc ggtgtaggt cgtt eget ccaagctgggctgtgtg cacgaaccccccgtt cagcccgaccgctgcgcct tatccggtaactatcgtcttgagtccaacccggt aagacacgacttatcgccactggcagcagccact ggtaacaggattagcagagcgaggtatgtaggcg gtgctacagagttcttgaagtggtggcctaacta cggctacactagaagaacagtatttggtatctgc get ct get gaagccagtt acct teggaaaaagag ttggtagctcttgatccggcaaacaaaccaccgc tggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcAttorney Docket No. GMV0005-401-PCctttgatctttt ctacggggtctgacgct cagtg gaacgaaaactcacgttaagggattttggtcatg agattatcaaaaaggatcttcacctagatccttt taaattaaaaatgaagttttaaatcaatctaaag tatatatgagtaaacttggtctgacagttaccaa tgcttaatcagtgaggcacctatctcagcgatct gtctatttcgttcatccatagttgcctgactccc cgtcgtgtagataactacgatacgggagggctta ccatctggccccagtgctgcaatgataccgcgag acccacgct caccggctccagatttat cagcaat aaaccagccagccggaagggccgagcgcagaagt ggtcctgcaactttatccgcctccatccagtcta ttaattgttgccgggaagctagagtaagtagtt c gccagttaatagtttgcgcaacgttgttgccatt gctacaggcatcgtggtgt cacgct cgtcgtttg gtatggctt catt cagct ccggttcccaacgat c aaggcgagttacatgatcccccatgttgtgcaaa aaagcggttagct ccttcggtcctccgat cgttg tcagaagtaagttggccgcagtgt tat cast cat ggttatggcagcactgcataattctcttactgtc atgccatccgtaagatgctttt ctgtgactggtg agtact caaccaagt catt ctgagaatagtgtat gcggcgaccgagttgctcttgcccggcgtcaata cgggataataccgcgccacatagcagaactttaa aagtgctcatcattggaaaacgttctt cggggcg aaaactctcaaggatcttaccgctgttgagatcc agttcgatgtaacccact cgtgcacccaactgat ctt cagcat cttttacttt caccagcgtttctgg gtgagcaaaaacaggaaggcaaaatgccgcaaaa aagggaataagggcgacacggaaatgttgaatac t catactcttcctttttcaatattattgaagcat ttatcagggttattgtctcatgagcggatacata tttgaatgtatttagaaaaataaacaaatagggg tt ccgcgcacatttccccgaaaagtgccacctga cgtctaagaaaccattattatcatgacattaacc tataaaaataggcgtatcacgaggccctttcgt c Lpxll upstreamSEQ ID NO: 37Forward ID22 GTCGGCAAGGGTAAGGCAGCLpxll upstreamSEQ ID NO: 38Reverse ID22 CCCTTTATACCGGCTGTCCGLpxlldownstreamSEQ ID NO: 39Forward ID22(720bp) ACTCAAAGCAGATGAAAAATCACLpxllSEQ ID NO: 40 downstreamReverse ID22 atatt cataggtttgcggtattt cAttorney Docket No. GMV0005-401-PCctgcaggccgtctgaaccgctctaaccgcttttt ctcggcttaatttttctgtctctgttataaaatt gctattcatcttgttcttcttcaaaaaaaagtta agtaaaat acct acct aaattttt act agttcgc aatctacgagcttataacctcgttttttcaattc atttaaaaaatcagattttgagcctaattttgat ctattgctatcgttacccgctagaaatacccagt aattacgcaaatcttcattggtaactttcgtaat atcggtgtaatgatcttcgagtatttttaagcaa t ct ctagcccataaaccgtact cgtgattgct ca tcttagggttttgcttatcgagtttgacgaactt cccatacttgtttttatgtggaaatactggccgt tttgcaacttctt caattttttgagctgttcgtt ttttactaccaatcacaaaatttaaagagtgaat agtacgcccacgcttgatttgtt caacct caacg actaaatcagatttct cgttaat ct cagttattg caggttccaaaacacgttgatttaatgaattaaa t ctagggtatt tatttt caacct gaagccatt ct tttagtttttctactgtaattt cacgactaccaa cagagcgatattgtgtaattagctcataaattcg aattgaatgtacactgttgaaataagcgatatgt ttgagttgatattgcgtgaattgccctttaagtt gcgttaggtatggcataacttcatcagtcattgc aattctaaaacgcccctctttcttgaaatatgtt ctagaggaaacccaacgaaatt cagttacacggt ctttatcttcagttttaacacttcggtcataaat ccgttttatagccgcctgaatttgcttataggcg pFP12-FHbp ID9SEQ ID NO: 41SM ttatcttggcttattt ctggaaact cacggacaa aatcagccaccgtaaaatcaaaaatcttttgatt agattt cggatccatagt cccaatagttaaagct aaaattctgatttcatcaatactcaatcggtaat tggcttcaataaggctattagcctttacaacaac taaat catttggcataagacaacaaattt cctgt ttaaaacaacaagcaaaatatacctgttgtttat atataaaacaacaagtattttcttaaaagttgtc tataacaggaaatttgttgt cttataacaggaaa tttgttgt cgtataacaggaaatttgttgtcgta taacaggaaatttgttgtcgtataagtttgtaac ttattgattttactggttttaaaaacgccgaaaa caagtaaaaaacaaaaatataaaaatatagggac tttcgtcccttttttgggctttcagccctaattt ttt ctttttttcaggatttaaaattacaaaaccc ttacagagcaagtaaacttgtttgcttgttctgc aagggttcagcaaccgtagccgtcaggcgtaggg cggtagcctataaaagccatttaattttatcttt aaactt ccttt taaat get ttgagtgggtgtctt ttatcgtactcatcaatccttttttgcattcttt cgtttgctttgtgat cggcaaattttgaataaga ttttt ccat ctcatctaacatt ctat caatccgt tttttatgttgccatttcaggtaaacataaacac ttatagcaattaaagacaatat caatacattgta aaaaatgattgttacaatttcgctcacagttatt ttttacctttttcaatttcttcattgataaatgc actcaattcatcaaatttcttgtcatcattgataaatttacgcaacttagggaagtttctatctacatAttorney Docket No. GMV0005-401-PCctaaaagagggttatttattatttcatttagcca aaaagcccctaataaaaccttgtaatgcgtagct ttcttacgcttttctgcttgttcttttgacttaa t cgcacgaatttt cgctttgatttcgt cctgctt gcgttgtaaatctgcttgttgctgttccaatctt gtaagtttttcgcttgccatactagcccctttat atagttagaaattat cgttattttatt cagtagg tgctaggcttgcaagtgttctgttcattacgtta aaataacgtaatgcccacttatcagtttctcttc gagaaactggtgggcaagcgtaccgcttgaccgt ttcgcaatactcaacactatggcaatctatcatt taaacgttcgctattgcagtaaaagcaaagggca at cagctcaagccaaaaacgactacat caaccgc aatgataaatattcaaagcggttagatgatttac agtttt caggctatggtaatatgccaaaatttgc cgaagataatccgcaagaattttggcgattgt ca gatatttacgagcgagctaatgcccgagtttgta ctgaaattgaatttgctttacctagagaattaac cctagaacaacagcaaaaattagtaagtt cgttt atagaaaatacggttgatagcggtagcaataaac taccctact cttt cgctat ccataccgataaaaa taatcataatccccattgt catttgatatttt ca gaacgccaacttgacggcatagaccgtacagccg agcagttttttaaacgtgctaatactaaatcccc agaaaagggcggagcgatgaaaacggcagatttt cgagatcgtgagtttatccaatctgtccgaaaaa cgtggagagagcaagctaat caagccttagagca atacggatatgccgcacgaattgacgaacgtagc tacaaggaacaaggcatagagcaagccccaagag caagaattgacagggtaacgtggcaagaattgaa ccgattagagcaagaagaacgccaaat cgtgcaa gagcttgcacttaaaggacaagaaattaacaaag aaaaat cctacttgcagaaaat cgaagaaaaaca ggctcaaggaatgggcaaatatgaat ccaaatt c gcagctgcgttttctaaattatcggaaagtgccc taaaacacgatttaagcaacgaaaaagaaaaaga cagtaaaatacacact caagaagaaaaagtgcct caaaatcgcattcaggggctttctcaagcagatt ttgat cagtttt taatt gat gaatggct acct ca aatagaaaaatacgttaaagcccaagaaaagcgg gacggaatggaagtagagatcacgcaatacgaca aggatttacagcgtattcagggagactataacaa get cacagataaaaat cagggtttt ct cggttta tgggaaactaaagagcaaaaagcaaagaaaaaag agcttgaagatgaatacaaacatacagcagagca acggaacgctaaaagccaagaattagccgagtat agccaaaaaataaaagcatacgaacagaaaacgc tagagccaatcaacgagaagattgccaaatat ca agctgacaaccctgaaat aaaaat geggagett a ggatttgtgaaaaaaattaaggctcaaggggcat ataaagcggctcaagagcgaatggagcgagaaaa acagcaccaacaggaaaaacaacagagacattta gagcgagagagtggtttgagcttgtagctaacgc cctacgcctacggctt cggttgttcaacccttaaagaactcgcaacaagttgcaaattctttaagggtAttorney Docket No. GMV0005-401-PCt cgcaataaaaacaaccgctaaacatttctgccc agcggttgaaaatttacctattcaccattacaat gatcaagcaggaaatttttttgattgccgtaaat gt ccgtatatctagttgaggcacaacccgccaaa gtcattgccccaaccagaacggcgaagctaatac gcaaaccgcctctccccgcgcgttggccgattca ttaatgcagctggcacgacaggttt cccgactgg aaagcgggcagtgagcgcaacgcaattaatgtga gttagctcactcattaggcaccccaggctttaca ctttatgcttccggct cgtatgttgtgtggaatt gtgagcggataacaatttcacacaggaaacagct atga CcatgattacgccaagcttCCTCAGCGCATGCAT CCGGGCAGCAGCGCATCGGCTCGCACGAGGTCTG CGCTTGAATTGTGTTGTAGAAACACAACGTTTTT G AAAAAAT AAG CTATTGTTTTATAT C AAAAT AT A AT C AT T T T T AAAAT AAAGGT T GC GGC AT T T AT C A GATATTTGTTCTGAAATGAAGACGTATCGGGTGT TTGCCCGATGTTTTTAGGTTTTTATCAAATTTAC AAAAGGAAGCCCATATGGTGAACCGAACTGCCTT CTGCCTTTCTCTGACCGCCGCCCTGATTCTGACC GCCTGCAGTAGCGGAGGCGGCGGAAGCGGAGGCG GCGGTGTCGCCGCCGACATCGGTGCGGGGCTTGC TGATGCACTAACCGCACCGCTCGACCATAAAGAC AAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCG T CAGGAAAAACGAGAAACT GAAGCT GGCGGC ACA AGGTGCGGAAAAAACTTATGGAAACGGCGACAGC CTCAATACGGGCAAATTGAAGAACGACAAGGTCA GCCGCTTCGACTTTATCCGTCAAATCGAAGTGGA CGGGCAGCTCATTACCTTGGAGAGCGGAGAGTTC CAAGTGTACAAACAAAGCCATTCCGCCTTAACCG CCCTTCAGACTGAGCAAGTACAAGACTCGGAGGA TTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGA ATCGGCGACATAGCGGGCGAACATACGTCTTTTG ACAAGCTTCCCAAAGGCGGCAGTGCGACATATCG CGGGACGGCGTTCGGTTCAGACGATGCTGGCGGA AAACTGACCTATACTATAGATTTCGCCGTCAAAC AGGGACACGGCAAAATCGAACATTTGAAATCGCC CGAACTCAATGTCGACCTGGCCGCCGCCTATATC AAGCCGGATAAAAAACGCCATGCCGTCATCAGCG GTTCCGTCCTTTACAACCAAGACGAGAAAGGCAG TTACCGCCTCGGCATCTTTGGCGGGCAAGCCCAG GAAGTTGCCGGCAGCGCGGAAGTGGAAACCGCAA ACGGCATACACCTTATCGGTCTTGCCGCCAAGCA GTAAAGGCCTCCTGCAGGactagtcggccgtacg ggcccttt cgtct cgcgcgttt cggtgattaagt gcggtcatcttcggtttccgtgtttcgtaaagtc tggaaacgcggaagt cccctacgtgctgctgaag ttgccccaacagagagtggaaccaaccggtgata ccacgatactatgactgagagtcaacgccatgag cggcct catttcttattctgagttacaacagt cc gcaccgctgtccggtagctccttccggtgggcgc ggggcatgactatcgtcgccgcacttatgactgt ctt ctttat catgcaact cgtaggacaggtgccggcagcgcccaacagtcccccggccacggggcctgAttorney Docket No. GMV0005-401-PCccaccatacccacgccgaaacaagcgccctgcac cattatgttccggatctgcatcgcaggatgctgc tggctaccctgtggaacacctacatctgtattaa cgaagcgctaaccgtttttatcaggct ctgggag gcagaataaatgatcatatcgtcaattattacct ccacggggagagcctgagcaaactggcctcaggc atttgagaagcacacggt cacactgcttccggta gtcaataaaccggtaaaccagcaatagacataag cggctatttaacgaccctgccctgaaccgacgac cgggt cgaatttgctttcgaatttctgccatt ca tccgcttattatcacttattcaggcgtagcacca ggcgtttaagggcaccaataactgccttaaaaaa attacgccccgccctgccactcatcgcagtactg ttgtaattcattaagcattctgccgacatggaag ccatcacagacggcatgatgaacctgaat cgcca gcggcatcagcaccttgt cgccttgcgtataata tttgcccatggtgaaaacgggggcgaagaagttg t ccatattggccacgtttaaat caaaactggtga aactcacccagggattggctgagacgaaaaacat attctcaataaaccctttagggaaataggccagg ttttcaccgtaacacgccacatcttgcgaatata tgtgtagaaactgccggaaatcgtcgtggtatt c actccagagcgatgaaacgtttcagtttgctcat ggaaaacggtgtaacaagggtgaacactatccca tatcaccagctcaccgtctttcattgccatacgg aattccggat gagcat t cat caggcgggcaagaa tgtgaataaaggccggataaaacttgtgcttatt ttt ctttacggt ctttaaaaaggccgtaatat cc agctgaacggtctggttataggtacattgagcaa ctgactgaaatgcct caaaatgttctttacgatg ccattgggatatatcaacggtggtatatccagtg atttttttctccattttagcttccttagctcctg aaaat etc gat aa ct caaaaaatacgcc eggtag tgatcttatttcattatggtgaaagttggaacct cttacgtgccgatcaacgtctcattttcgccaaaagttggcccagggctt cccggtatcaacaAttorney Docket No. GMV0005-401-PCCTGCAGGCCGTCTGAACCGCTCTAACCGCTTTTT CTCGGCTTAATTTTTCTGTCTCTGTTATAAAATT GCTATTCATCTTGTTCTTCTTCAAAAAAAAGTTA AGTAAAATACCTACCTAAATTTTTACTAGTTCGC AATCTACGAGCTTATAACCTCGTTTTTTCAATTC ATTTAAAAAATCAGATTTTGAGCCTAATTTTGAT CTATTGCTATCGTTACCCGCTAGAAATACCCAGT AATTACGCAAATCTTCATTGGTAACTTTCGTAAT ATCGGTGTAATGATCTTCGAGTATTTTTAAGCAA TCTCTAGCCCATAAACCGTACTCGTGATTGCTCA TCTTAGGGTTTTGCTTATCGAGTTTGACGAACTT CCCATACTTGTTTTTATGTGGAAATACTGGCCGT TTTGCAACTTCTTCAATTTTTTGAGCTGTTCGTT T T T T AC T AC CAAT C ACAAAAT T T AAAGAGT GAAT AGTACGCCCACGCTTGATTTGTTCAACCTCAACG ACTAAATCAGATTTCTCGTTAATCTCAGTTATTG C AGGT T C C AAAAC AC GT T GAT T T AAT GAAT T AAA TCTAGGGTATTTATTTTCAACCTGAAGCCATTCT TTTAGTTTTTCTACTGTAATTTCACGACTACCAA CAGAGCGATATTGTGTAATTAGCTCATAAATTCG AATTGAATGTACACTGTTGAAATAAGCGATATGT TTGAGTTGATATTGCGTGAATTGCCCTTTAAGTT GCGTTAGGTATGGCATAACTTCATCAGTCATTGC AATTCTAAAACGCCCCTCTTTCTTGAAATATGTT C T AGAGG AAAC C C AAC GAAAT T C AG T T AC AC G GT CTTTATCTTCAGTTTTAACACTTCGGTCATAAAT CCGTTTTATAGCCGCCTGAATTTGCTTATAGGCGpFP12-FHbpSEQ ID NO: 42 TTATCTTGGCTTATTTCTGGAAACTCACGGACAA ID22 TMAAT CAGC C ACC GT AAAAT CAAAAAT C T T T T G AT T AGATTTCGGATCCATAGTCCCAATAGTTAAAGCT AAAAT T C T GAT T T C AT CAAT AC T CAAT C GGT AAT T GGCT T C AATAAGGC T AT T AGC C T T T AC AAC AAC T AAAT C AT T T G GC AT AAG AC AAC AAAT T T CC T GT T T AAAACAACAAGCAAAAT AT ACCT GT TGTT T AT AT ATAAAACAACAAGT AT T T TCT TAAAAGTT GT C TATAACAGGAAATTTGTTGTCTTATAACAGGAAA TTTGTTGTCGTATAACAGGAAATTTGTTGTCGTA TAACAGGAAATTTGTTGTCGTATAAGTTTGTAAC T T ATT GAT T T T AC T G GT T T T AAAAAC GC C GAAAA CAAGTAAAAAACAAAAATATAAAAATATAGGGAC TTTCGTCCCTTTTTTGGGCTTTCAGCCCTAATTT TTTCTTTTTTTCAGGATTTAAAATTACAAAACCC TTACAGAGCAAGTAAACTTGTTTGCTTGTTCTGC AAGGGTTCAGCAACCGTAGCCGTCAGGCGTAGGG CGGTAGCCTATAAAAGCCATTTAATTTTATCTTT AAACTTCCTTTTAAATGCTTTGAGTGGGTGTCTT TTATCGTACTCATCAATCCTTTTTTGCATTCTTT CGTTTGCTTTGTGATCGGCAAATTTTGAATAAGA TTTTTCCATCTCATCTAACATTCTATCAATCCGT TTTTTATGTTGCCATTTCAGGTAAACATAAACAC TTATAGCAATTAAAGACAATATCAATACATTGTA AAAAATGATTGTTACAATTTCGCTCACAGTTATT TTTTACCTTTTTCAATTTCTTCATTGATAAATGC ACTCAATTCATCAAATTTCTTGTCATCATTGATAAATTTACGCAACTTAGGGAAGTTTCTATCTACATAttorney Docket No. GMV0005-401-PCC T AAAAG AGGG TTATTTATTATTTCATTTAGCCA AAAAGCCCCTAATAAAACCTTGTAATGCGTAGCT TTCTTACGCTTTTCTGCTTGTTCTTTTGACTTAA TCGCACGAATTTTCGCTTTGATTTCGTCCTGCTT GCGTTGTAAATCTGCTTGTTGCTGTTCCAATCTT GTAAGTTTTTCGCTTGCCATACTAGCCCCTTTAT AT AGT T AG AAAT T AT CGTTATTTTATTCAGTAGG TGCTAGGCTTGCAAGTGTTCTGTTCATTACGTTA AAATAACGTAATGCCCACTTATCAGTTTCTCTTC GAGAAACTGGTGGGCAAGCGTACCGCTTGACCGT T T CGCAAT ACT CAACACT AT GGCAAT CT ATCAT T TAAACGTTCGCTATTGCAGTAAAAGCAAAGGGCA AT C AG C T C AAG C C AAAAAC GAC T AC AT C AAC C GC AAT GAT AAATAT T CAAAGCGGT T AGAT GATT T AC AGTTTTCAGGCTATGGTAATATGCCAAAATTTGC CGAAGATAATCCGCAAGAATTTTGGCGATTGTCA GATATTTACGAGCGAGCTAATGCCCGAGTTTGTA CTGAAATTGAATTTGCTTTACCTAGAGAATTAAC CCTAGAACAACAGCAAAAATTAGTAAGTTCGTTT ATAGAAAATACGGTTGATAGCGGTAGCAATAAAC TACCCTACTCTTTCGCTATCCATACCGATAAAAA T AAT C AT AAT C C C C AT T G T C AT T T G AT AT T T T C A GAACGCCAACTTGACGGCATAGACCGTACAGCCG AGCAGTTTTTTAAACGTGCTAATACTAAATCCCC AGAAAAGGGCGGAGCGATGAAAACGGCAGATTTT CGAGATCGTGAGTTTATCCAATCTGTCCGAAAAA CGTGGAGAGAGCAAGCTAATCAAGCCTTAGAGCA ATACGGATATGCCGCACGAATTGACGAACGTAGC TACAAGGAACAAGGCATAGAGCAAGCCCCAAGAG CAAGAATTGACAGGGTAACGTGGCAAGAATTGAA CCGATTAGAGCAAGAAGAACGCCAAATCGTGCAA GAGCT T GCACT TAAAGGACAAGAAAT T AACAAAG AAAAAT C C T AC T T GC AGAAAAT C GAAGAAAAAC A GGCTCAAGGAATGGGCAAATATGAATCCAAATTC GCAGCTGCGTTTTCTAAATTATCGGAAAGTGCCC T AAAACAC GAT T T AAGCAACGAAAAAGAAAAAGA C AGT AAAAT AC AC AC T CAAGAAGAAAAAGT GC C T CAAAATCGCATTCAGGGGCTTTCTCAAGCAGATT TTGATCAGTTTTTAATTGATGAATGGCTACCTCA AATAGAAAAATACGTTAAAGCCCAAGAAAAGCGG GACGGAATGGAAGTAGAGATCACGCAATACGACA AGGATTTACAGCGTATTCAGGGAGACTATAACAA GCTCACAGATAAAAATCAGGGTTTTCTCGGTTTA TGGGAAACT7AAGAGC7AAAAGC7AAGAAAAAAG AGCTTGAAGATGAATACAAACATACAGCAGAGCA ACGGAACGCTAAAAGCCAAGAATTAGCCGAGTAT AGCCAAAAAATAAAAGCATACGAACAGAAAACGC TAGAGCCAATCAAC G AGAAGAT TGC C AAAT AT C A AGC T GAC AACC C T GAAATAAAAAT GC GG AGC T T A GGATTTGTG7AAAAAATTAAGGCTCAAGGGGCAT ATAAAGCGGCTCAAGAGCGAATGGAGCGAGAAAA ACAGCACCAACAGGAAAAACAACAGAGACAT T TA GAGCGAGAGAGTGGTTTGAGCTTGTAGCTAACGC CCTACGCCTACGGCTTCGGTTGTTCAACCCTTAAAGAACTCGCAACAAGTTGCAAATTCTTTAAGGGTAttorney Docket No. GMV0005-401-PCT C GC AAT AAAAAC AAC CG C T AAACAT T T C T GC C C AGCGGTTGAAAATTTACCTATTCACCATTACAAT GATCAAGCAGGAAATTTTTTTGATTGCCGTAAAT GTCCGTATATCTAGTTGAGGCACAACCCGCCAAA GTCATTGCCCCAACCAGAACGGCGAAGCTAATAC GCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCA TTAATGCAGCTGGCACGACAGGTTTCCCGACTGG AAAGCGGGCAGTGAGCGCAACGCAATTAATGTGA GTTAGCTCACTCATTAGGCACCCCAGGCTTTACA CTTTATGCTTCCGGCTCGTATGTTGTGTGGAATT GTGAGCGGATAACAATTTCACACAGGAAACAGCT ATGACCATGATTACGCCAAGCTTCCTCAGCGCAT GCATCCGGGCAGCAGCGCATCGGCTCGCACGAGG TCTGCGCTTGAATTGTGTTGTAGAAACACAACGT T T T TGAAAAAAT AAGCTAT T GT T TT AT AT CAAAA TATAATCATTTTTAAAATAAAGGTTGCGGCATTT ATCAGATATTTGTTCTGAAATGAAGACGTATCGG GTGTTTGCCCGATGTTTTTAGGTTTTTATCAAAT T T AC AAAAGGAAGC C C AT AT GG T GAAC C GAAC T G CCTTCTGCCTTTCTCTGACCGCCGCCCTGATTCT GACCGCCTGCAGTAGCGGAGGCGGCGGAAGCGGA GGCGGCGGTGTCGCCGCCGACATCGGTGCGGGGC TTGCTGATGCACTAACCGCACCGCTCGACCATAA AGACAAAGGTTTGCAGTCTTTGACGCTGGATCAG TCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGG CACAAGGTGCGGAAAAAACTTATGGAAACGGCGA CAGCCTCAATACGGGCAAATTGAAGAACGACAAG GTCAGCCGCTTCGACTTTATCCGTCAAATCGAAG TGGACGGGCAGCTCATTACCTTGGAGAGCGGAGA GTTCCAAGTGTACAAACAAAGCCATTCCGCCTTA ACCGCCCTTCAGACTGAGCAAGTACAAGACTCGG AGGATTCCGGGAAGATGGTTGCGAAACGCCAGTT CAGAATCGGCGACATAGCGGGCGAACATACGTCT TTTGACAAGCTTCCCAAAGGCGGCAGTGCGACAT ATCGCGGGACGGCGTTCGGTTCAGACGATGCTGG CGGAAAACTGACCTATACTATAGATTTCGCCGTC AAACAGGGACACGGCAAAATCGAACATTTGAAAT CGCCCGAACTCAATGTCGACCTGGCCGCCGCCTA TATCAAGCCGGATAAAAAACGCCATGCCGTCATC AGCGGTTCCGTCCTTTACAACCAAGACGAGAAAG GCAGTTACCGCCTCGGCATCTTTGGCGGGCAAGC CCAGGAAGTTGCCGGCAGCGCGGAAGTGGAAACC GCAAACGGCATACACCTTATCGGTCTTGCCGCCA AGCAGTAAAGGCCTCCTGCAGGACTAGTCGGCCG TACCTGTCAGACCAAGTTTACTCATATATACTTT AGAT T GAT T T AAAAC T T C AT T T T T AAT T T AAAAG GATCTAGGTGAAGATCCTTTTTGATAATCTCATG ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACT GAGCGTCAGACCCCGTAGAAAAGATCAAAGGATC TTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGC TGCTTGCAAACAAAAAAACCACCGCTACCAGCGG TGGTTTGTTTGCCGGATCAAGAGCTACCAACTCT TTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAG ATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCAttorney Docket No. GMV0005-401-PCTACATACCTCGCTCTGCTAATCCTGTTACCAGTG GCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG GGTTGGACTCAAGACGATAGTTACCGGATAAGGC GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA CAGCCCAGCTTGGAGCGAACGACCTACACCGAAC TGAGATACCTACAGCGTGAGCTATGAGAAAGCGC CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCT TTATAGTCCTGTCGGGTTTCGCCACCTCTGACTT GAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTT TTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT CACATGTGGGCCCTTTCGTCTCGCGCGTTTCGGT GATTAAGTGCGGTCATCTTCGGTTTCCGTGTTTC GTAAAGTCTGGAAACGCGGAAGTCCCCTACGTGC TGCTGAAGTTGCCCCAACAGAGAGTGGAACCAAC CGGTGATACCACGATACTATGACTGAGAGTCAAC GCCATGAGCGGCCTCATTTCTTATTCTGAGTTAC AACAGTCCGCACCGCTGTCCGGTAGCTCCTTCCG GTGGGCGCGGGGCATGACTATCGTCGCCGCACTT ATGACTGTCTTCTTTATCATGCAACTCGTAGGAC AGGTGCCGGCAGCGCCCAACAGTCCCCCGGCCAC GGGGCCTGCCACCATACCCACGCCGAAACAAGCG CCCTGCACCATTATGTTCCGGATCTGCATCGCAG GATGCTGCTGGCTACCCTGTGGAACACCTACATC TGTATTAACGAAGCGCTAACCGTTTTTATCAGGC TCTGGGAGGCAGAATAAATGATCATATCGTCAAT TATTACCTCCACGGGGAGAGCCTGAGCAAACTGG CCTCAGGCATTTGAGAAGCACACGGTCACACTGC TTCCGGTAGTCAATAAACCGGTAAACCAGCAATA GACATAAGCGGCTATTTAACGACCCTGCCCTGAA CCGACGACCGGGTCGAATTTGCTTTCGAATTTCT GCCATTCATCCGCTTATTATCACTTATTCAGGCG TAGCACCAGGCGTTTAAGGGCACCAATAACTGCC TTAAAAAAATTACGCCCCGCCCTGCCACTCATCG CAGTACTGTTGTAATTCATTAAGCATTCTGCCGA CATGGAAGCCATCACAGACGGCATGATGAACCTG AATCGCCAGCGGCATCAGCACCTTGTCGCCTTGC GTATAATATTTGCCCATGGTGAAAACGGGGGCGA AGAAGTTGTCCATATTGGCCACGTTTAAATCAAA ACTGGTGAAACTCACCCAGGGATTGGCTGAGACG AAAAAC AT AT T C T C AATAAAC C C T T T AGGGAAAT AGGCCAGGTTTTCACCGTAACACGCCACATCTTG CGAATATATGTGTAGAAACTGCCGGAAATCGTCG TGGTATTCACTCCAGAGCGATGAAACGTTTCAGT TTGCTCATGGAAAACGGTGTAACAAGGGTGAACA CTATCCCATATCACCAGCTCACCGTCTTTCATTG CCATACGGAATTCCGGATGAGCATTCATCAGGCG GGCAAGAATGTGAATAAAGGCCGGATAAAACTTG TGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCG TAATATCCAGCTGAACGGTCTGGTTATAGGTACA TTGAGCAACTGACTGAAATGCCTCAAAATGTTCT TTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAttorney Docket No. OMV0005-401-PCCharacterization of mutant of Nm strain H44 / 76 containing 3 chromosomal copies coding for FHbp ID9 SM or FHbp ID22 TM and a multi-copy plasmid coding for FHbp ID9 SM or FHbp ID22 TM,PCR

[0097] PCR primers were designed in order to amplify upstream and downstream the constructs inserted in Neisseria meningitidis strain H44 / 76 carrying the flanking region for the flibp, siaD-galE, or IpxLl genes, the FHbp ID9 SM or FHbp ID22 TM gene, and the antibiotic resistant cassette. PCR was performed on heat killed cells. The heat killed cells from the wild-type H44 / 76 were used as negative control.Flow cytometry

[0098] Binding of purified monoclonal antibodies against FHbp ID9 SM or FHbp ID22 TM to the surface of live N. meningitidis bacteria was measured by flow cytometry asAttorney Docket No. OMV0005-401-PCdescribed previously. H44 / 76, engineered to expresses the target antigens, was used as the test strain. Briefly, bacteria were grown in Frantz+lactate or chemically defined medium (CDM) containing 20mM instead of 4mM lactate, up to an OD620nm of 0.6-0.7. To measure anti-ID9 or anti-ID22 antibody binding, a fixed concentration of JAR5 or JAR32 antibodies or, as a negative control, 10 pg / mL of an irrelevant antibody, was incubated with 107bacteria / niL. Bound antibody was detected using AlexaFluor 488-conjugated goat anti-mouse or rabbit IgG secondary antibody (Jackson Immuno Research Laboratories). FHbp expression on H44 / 76 strains engineered to express three chromosomal copies of FHbp ID22 TM or ID9 SM with the multi copy plasmid pFP12 is shown in Table 5. FHbp expression was determined by binding of monoclonal antibodies JAR5 and JAR32 specific for subfamily B and subfamily A FHbp respectively. The data shows increased median fluorescence intensity, resulted by enhanced binding enhanced binding by flow cytometry of anti-FHbp polyclonal antibodies to the lab passaged strain of H44 / 76 lacking PorA in which siaD-galE, IpxLl and fhbp loci have been disrupted with copies of genes coding for FHbp ID9 SM and FHbp ID22 TM, respectively, and, in addition, carrying a multi -copy plasmid (see Table 5) with each respective gene.Table 5Median Fluorescence Intensity (MFI)ID22ID9 SMTMParent Strain 39 54271 copy 1446 57032 copies 8184 81843 copies 18264 190943 copies + pFP12 31016 39719Irrelevant antibody 44 20.5Example 2

[0099] Knocking out IpxLl and rmpM genes in N. gonorrhoeae (Ng) strain.Transformation of N.gonorrhoeae.

[0100] The N. gonorrheae strains FA 1090, WHO F, WHO M and WHO N in which the IpxLl and rmpM genes were inactivated (FA 1090 Armp AlpxLl, WHO F Armp AlpxLl, WHO M Armp AlpxLl, and WHO N Armp AlpxLl respectively) were made by homologous recombination by transformation with plasmids pUC18-lpxLlKO-CAT using chloramphenicol selection (2.5 pg / ml), and pUC18-rmpMKO-KAN using kanamycinAttorney Docket No. GMV0005-401-PCselection (40 pg / ml). Transformations starting from the wild-type strain were carried in the following order:(1) The rmpM gene was knocked out with pUC18-rmpMKO-KAN plasmid.(2) The IpxLl gene was knocked out with pUC18-lpxLlKO-CAT plasmid.

[0101] Ten to 15 colonies of each strain were selected from a Chocolate II Agar plates (Remel, Thermo Fisher Scientific, Pittsburgh, PA) that had been grown overnight. The colonies of bacteria were mixed with 1-3 pg of the plasmid, plated onto a Chocolate II agar plate, and incubated for 6 hrs at 37°C. Serial dilutions of the bacteria were re-cultured onto Chocolate II agar plates containing antibiotic for selection. The culture plates were incubated overnight at 37°C, and the colonies were screened for the lack of expression of IpxLl and rmpMby PCR using heat killed cells. Positive individual colonies were frozen in 10% skim milk (wt / vol) and 15% glycerol, and stored at -80°C. Plasmids pUC18-lpxLlKO-CAT and pUC18-rmpMKO-KAN are shown in Table 6.Table 6.Identifier Protein SequenceAttorney Docket No. GMV0005-401-PCtcgcgcgtttcggtgatgacggtgaaaacctctgac acatgcagctcccggagacggtcacagcttgtctgt aagcggatgccgggagcagacaagcccgtcagggcg cgtcagcgggtgttggcgggtgtcggggctggctta actatgcggcatcagagcagattgtactgagagtgc accatatgcggtgtgaaataccgcacagatgcgtaa ggagaaaataccgcatcaggcgccattcgccattca ggctgcgcaactgttgggaagggcgatcggtgcggg cctctt cgctattacgccagctggcgaaagggggat gtgctgcaaggcgattaagttgggtaacgccagggt tttcccagtcacgacgttgtaaaacgacggccagtg CCaagcttATGCCGATTTCTTCTTCOAGCAATTTGA TTTGTTTGGAGATGCCGGGTTGTGATGTGAATAAGG CTTCGGCCGCTTCGGAAACGTTCAGGTTGTGCCGGT AAACTTCTAAGGCGTATTTCAATTGTTGTAATTTCA TGGCGGGTCGGTGTGGGTCTGTGTCGGGTGGCTGAA CATTGTTTATAATTTATCATATTTTCTTGCCGGTAC GGTATGGGGCTTTGCCGTTGTGTTTGTTGTTTTTGT G C AAC G G C AAT C G T G C G AT AT G GAAAAAAT C C C C C T AAAGTAATGACACGGAATTGATTTTTCGGCATGATA GACTATCAGGAAACAGGCTGTTTTACGGTTGTTTTC AGGCGTTGAGTATTGACAGTCCGCCCCCTGTTTCTT TATAGTGGAGACT G AAAT AT CCGATTTGCCGCCATGpUC18- TTTCTACAGCGGCCTGTATGTTGGCAATTCAGCAGTrmpMKO- TGCTTCTGTATCTGCTGTACAAATCTAATGAGGGAA KAN SEQ ID NO: 43 TAAAATGACCAAACAGCTGGCATTGAGCTAGTCAGT plasmidGAG GAG AAAT C GAT T AAT T AAAT G G C GAT AG C TAG Afull CTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTsequence GCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCC CTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAG GATCTGATGGCGCAGGGGATCAAATCTGATCAAGAG ACAGGATGAGGATCGTTTCGCATGATTGAACAAGAT GGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAG AGGCTATTCGGCTATGACTGGGCACAACAGACAATC GGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCG CAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTG TCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCG CGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGC GCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGG GACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGAT CTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTA TCCATCATGGCTGATGCAATGCGGCGGCTGCATACG CTTGATCCGGCTACCTGCCCATTCGACCACCAAGCG AAACATCGCATCGAGCGAGCACGTACTCGGATGGAA GCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAG CATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGG CTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTC GTGACCCATGGCGATGCCTGCTTGCCGAATATCATG GTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGT GGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCAttorney Docket No. GMV0005-401-PCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGT ATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTAT CGCCTTCTTGACGAGTTCTTCTGATTCAGACGGCAT GCGGCCGCCATCACCAACACTAAGGCTAGGTAATAT CTTGCCGATGCATGAGGTTAGCGGATTTTGTACCGG GTACTGTTGCAATATTCGTGAAACGTCGGCCGGTAT CGATGATGTGAAACAAACCCCGCTTTTGCGGGGTTTGTTTTTTTGGGTGGTTTTCTGAAACGGCTATCGTCAGAATCGGGGTGCAGGTTCGGATTCGGATTCAGATTC ATGTTTGTGTCCCATTGCCGCGCTTTATAGTGGATT AACAAAAATCAGGACAAGGCGACGAAGCCGCAGACA GTACAATAGTACGGCAAGGCGAGGCAACGCCGTACC GGTTTAAATTTAATCCACTATATCGGTTGAAACTCT GATTTTAAGGCGGTAGGATGTGGGTTTGCCCATAGC AAGGGAATCCTTTCTGTATCAAGCCCCGAAAGGGAT AATTCATACAAATTCACGCCTTTCCCCCTCATTGGG AAATGGATGGAATCGTGCCCGATGTGTGCGGCACTG TATGCCGGATATGGTTTTAtctagaggatccccggg taccgagctcgaattcgtaatcatggtcatagctgt ttcctgtgtgaaattgttatccgctcacaattccac acaacatacgagccggaagcataaagtgtaaagcct ggggtgcctaatgagtgagctaactcacattaattg cgttgcgctcactgcccgctttccagtcgggaaacc tgtcgtgccagctgcattaatgaatcggccaacgcg cggggagaggcggtttgcgtattgggcgctcttccg cttcctcgctcactgactcgctgcgctcggtcgttc ggctgcggcgagcggtatcagctcactcaaaggcgg taatacggttatccacagaatcaggggataacgcag gaaagaacatgtgagcaaaaggccagcaaaaggcca ggaaccgtaaaaaggccgcgttgctggcgtttttcc ataggctccgcccccctgacgagcatcacaaaaatc gacgctcaagtcagaggtggcgaaacccgacaggac tataaagataccaggcgtttccccctggaagctccc tcgtgcgctctcctgttccgaccctgccgcttaccg gatacctgtccgcctttctcccttcgggaagcgtgg cgctttctcaaagctcacgctgtaggtatctcagtt cggtgtaggtcgttcgctccaagctgggctgtgtgc acgaaccccccgttcagcccgaccgctgcgccttat ccggtaactatcgtcttgagtccaacccggtaagac acgacttatcgccactggcagcagccactggtaaca ggattagcagagcgaggtatgtaggcggtgctacag agttcttgaagtggtggcctaactacggctacacta gaagaacagtatttggtatctgcgctctgctgaagc cagttaccttcggaaaaagagttggtagctcttgat ccggcaaacaaaccaccgctggtagcggtggttttt ttgtttgcaagcagcagattacgcgcagaaaaaaag gatctcaagaagatcctttgatcttttctacggggt ctgacgctcagtggaacgaaaactcacgttaaggga ttttggtcatgagattatcaaaaaggatcttcacct agatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttAttorney Docket No. GMV0005-401-PCaccaatgcttaatcagtgaggcacctatctcagcga tctgtctatttcgttcatccatagttgcctgactcc ccgtcgtgtagataactacgatacgggagggcttac catctggccccagtgctgcaatgataccgcgagacc cacgctcaccggctccagatttatcagcaataaacc age cage cggaagggccgagcgcagaagtggtcctg caactttatccgcctccatccagtctattaattgtt gccgggaagctagagtaagtagttcgccagttaata gtttgcgcaacgttgttgccattgctacaggcatcg tggtgtcacgctcgtcgtttggtatggcttcattca gctccggttcccaacgatcaaggcgagttacatgat cccccatgttgtgcaaaaaagcggttagctccttcg gtcctccgatcgttgtcagaagtaagttggccgcag tgttatcactcatggttatggcagcactgcataatt ctcttactgtcatgccatccgtaagatgcttttctg tgactggtgagtactcaaccaagtcattctgagaat agtgtatgcggcgaccgagttgctcttgcccggcgt caatacgggataataccgcgccacatagcagaactt taaaagtgctcatcattggaaaacgttcttcggggc gaaaactctcaaggatcttaccgctgttgagatcca gttcgatgtaacccactcgtgcacccaactgatctt cagcatcttttactttcaccagcgtttctgggtgag caaaaacaggaaggcaaaatgccgcaaaaaagggaa taagggcgacacggaaatgttgaatactcatactct tcctttttcaatattattgaagcatttatcagggtt attgtctcatgagcggatacatatttgaatgtattt agaaaaataaacaaataggggttccgcgcacatttc cccgaaaagtgccacctgacgtctaagaaaccatta ttatcatgacattaacctataaaaataggcgtatca egaggee ct ttegtetcgcgcgtttcggtgatgacggtgaaaacctctgac acatgcagctcccggagacggtcacagcttgtctgt pUC18- aagcggatgccgggagcagacaagcccgtcagggcg IpxLKO- cgtcagcgggtgttggcgggtgtcggggctggctta CAT SEQ ID NO: 44 actatgcggcatcagagcagattgtactgagagtgc plasmid accatatgcggtgtgaaataccgcacagatgcgtaa full ggagaaaataccgcatcaggcgccattcgccattca sequenceggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatAttorney Docket No. GMV0005-401-PCgtgctgcaaggcgattaagttgggtaacgccagggt tttcccagtcacgacgttgtaaaacgacggccagtg ccaagcttgcatgcctgcaggtcgacGCCGTCTGAA AAG AAAAT G T G T AT C G AG AT G AAAT TTATATTTTTT GTACTGTATGTTTTGCAGTTTCTGCCGTTTGCGCTG CTGCACAAGATTGCCGACCTGACGGGTTTGCTTGCC TACCTTCTGGTCAAACCGCGCCGCCGTATCGGCGAA ATCAATTTGGCAAAATGTTTTTCCGAATGGAGTGAG GAAAAGC GT AAAAC C G T GT T G AAAC AGC AT T T C AAA CACATGGCGAAACTGATGTTGGAATACGGTTTATAT TGGTACGCGCCTGCCGGACGTTTGAAATCGCTGGTG CGCTACCGCAATAAGCATTATTTGGACGACGCGCTG GCGGCGGGGGAAAAAGTCATCATCCTGTATCCGCAC TTCACCGCTGCAGTTCTCGAGCTGCAGGTCATGCCG T C T G AAAAC C G T GAT AT AGAT T GAAAAGT GG AT AGA TTTATGATATAGTGGATAGATTTATGATATAATGAG T T AT C AAC AAAT C GGAATT T AC GGAG GAT AAAT GAT GCAATTCACAAAGATTGATATAAATAATTGGACACG AAAAG AG T AT T T C G AC C AC T AT T T T G GC AAT AC G C C CTGCACATATAGTATGACGGTAAAACTCGATATTTC T AAG T T G AAAAAG G AT G G AAAAAAG T T AT AC C C AAC TCTTTTATATGGAGTTACAACGATCATCAATCGACA T G AAG AG T T C AG G AC CGCATTAGAT G AAAAC G G AC A GGTAGGCGTTTTTTTCAGAAATGCTGCCTTGCTACA CAGTTTTT C AT AAGG AAAC T G AAAC CTTTTCGAGTA TTTGGACTGAGTTTACAGCAGACTATACTGAGTTTC TTCAGAACTATCAAAAGGATATAGACGCTTTTGGTG AAC GAAT G G G AAT GT C C G CAAAGC C TAAT C C T C C G G AAAACACTTTCCCTGTTTCTATGATACCGTGGACAA G C T T T G AAG G C T T T AAC T T AAAT C TAAAAAAAG GAT ATGACTATCTACTGCCGATATTTACGTTTGGGAAGT ATTATGAGGAGGGCGGAAAATACTATATTCCCTTAT CGATTCAAGTGCATCATGCCGTTTGTGACGGCTTTC ATGTTTGCCGTTTTTTGGATGAATTACAAGACTTGC TGAATAAATAAAATCCCAGTTTGTCGCACTGATAAA AACCCTTTAGGAACTAAAGGGCGCACTTCTATACTC TCTGTCGAGAGTAGTGCGTCCCTCAGCTGGAAGCCT GCAGGGAATAATGAGTCGACAGGATTTCGGACGCAA CGATTCGGTTTTTGTGGATTTTTTCGGTATTCAGAC GGCAACGATTACCGGATTGAGCCGCATTGCCGCGCT T GC AAAT GCAAAAGT G AT AC C C GC C ATT C C C GT C C G CGAGGCAGACAATACGGTTACATTGCATTTCTACCC TGCTTGGAAATCCTTTCCGGGTGAAGACGCGAAAGC CGACGCGCAGCGCATGAACCGTTTTATCGAAGACAG GGTGCGCGAACATCCGGAACAATATTTTTGGCTGCA CAAGCGTTTTAAAACCCGTCCGGAAGGCAGCCCCGA TTTTTACTGACTACGTCAGACGGCtctagaggatcc ccgggtaccgagctcgaattcgtaatcatggtcata gctgtttcctgtgtgaaattgttatccgctcacaat tccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattAttorney Docket No. GMV0005-401-PCaattgcgttgcgctcactgcccgctttccagtcggg aaacctgtcgtgccagctgcattaatgaatcggcca acgcgcggggagaggcggtttgcgtattgggcgctc ttccgcttcctcgctcactgactcgctgcgctcggt cgttcggctgcggcgagcggtatcagctcactcaaa ggcggtaatacggttatccacagaatcaggggataa cgcaggaaagaacatgtgagcaaaaggccagcaaaa ggccaggaaccgtaaaaaggccgcgttgctggcgtt tttccataggctccgcccccctgacgagcatcacaa aaatcgacgctcaagtcagaggtggcgaaacccgac aggactataaagataccaggcgtttccccctggaag ctccctcgtgcgctctcctgttccgaccctgccgct taccggatacctgtccgcctttctcccttcgggaag cgtggcgctttctcaaagctcacgctgtaggtatct cagttcggtgtaggtcgttcgctccaagctgggctg tgtgcacgaaccccccgttcagcccgaccgctgcgc cttatccggtaactatcgtcttgagtccaacccggt aagacacgacttatcgccactggcagcagccactgg taacaggattagcagagcgaggtatgtaggcggtgc tacagagttcttgaagtggtggcctaactacggcta cactagaagaacagtatttggtatctgcgctctgct gaagccagttaccttcggaaaaagagttggtagctc ttgatccggcaaacaaaccaccgctggtagcggtgg tttttttgtttgcaagcagcagattacgcgcagaaa aaaaggatctcaagaagatcctttgatcttttctac ggggtctgacgctcagtggaacgaaaactcacgtta agggattttggtcatgagattatcaaaaaggatctt cacctagatccttttaaattaaaaatgaagttttaa atcaatctaaagtatatatgagtaaacttggtctga cagttaccaatgcttaatcagtgaggcacctatctc agcgatctgtctatttcgttcatccatagttgcctg actccccgtcgtgtagataactacgatacgggaggg cttaccatctggccccagtgctgcaatgataccgcg agacccacgctcaccggctccagatttatcagcaat aaaccagccagccggaagggccgagcgcagaagtgg tcctgcaactttatccgcctccatccagtctattaa ttgttgccgggaagctagagtaagtagttcgccagt taatagtttgcgcaacgttgttgccattgctacagg catcgtggtgtcacgctcgtcgtttggtatggcttc attcagctccggttcccaacgatcaaggcgagttac atgatcccccatgttgtgcaaaaaagcggttagctc cttcggtcctccgatcgttgtcagaagtaagttggc cgcagtgttatcactcatggttatggcagcactgca taattctcttactgtcatgccatccgtaagatgctt ttctgtgactggtgagtactcaaccaagtcattctg agaatagtgtatgcggcgaccgagttgctcttgccc ggcgtcaatacgggataataccgcgccacatagcag aactttaaaagtgctcatcattggaaaacgttcttc ggggcgaaaactctcaaggatcttaccgctgttgag atccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctggAttorney Docket No. GMV0005-401-PCgtgagcaaaaacaggaaggcaaaatgccgcaaaaaa gggaataagggcgacacggaaatgttgaatactcat actcttcctttttcaatattattgaagcatttatca gggttattgtctcatgagcggatacatatttgaatg tatttagaaaaataaacaaataggggttccgcgcac atttccccgaaaagtgccacctgacgtctaagaaac cattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtcPCR

[0102] PCR primers were designed in order to amplify upstream and downstream the constructs inserted in Neisseria gonorrhoeae strain FA1090 carrying the flanking region for the rmpM, or IpxLl genes, and the antibiotic resistant cassette. PCR was performed on heat killed cells. The heat killed cells from the wild-type strains were used as negative controls. These primers are shown in Table 7.Table 7.Identifier Protein SequenceRmp Upstream KANSEQ ID NO: 45Forward CTGTTTTACGGTTGTTTTCAGGCGTTG Rmp Upstream KANSEQ ID NO: 46Reverse GGACAGGTCGGTCTTGACAAAAAGAACC Lpxl UpstreamSEQ ID NO: 47CAT Forward GTCGGCAAGGGTAAGGCAGCLpxl UpstreamSEQ ID NO: 48CAT Reverse CTTTGTGAATTGCATCATTTATCCTCCGExample 3

[0103] This example describes the preparation of chemically defined medium that supports the growth of Neisseria meningitidis bacteria to high densities needed for the production of native outer membrane vesicles (NOMV) that can be used as vaccines to elicit antibodies to recombinant antigens. In some embodiments, the chemically defined medium is described in Muller MG, Moe NE, Richards PQ, Moe GR. 2015. Resistance of Neisseria meningitidis to human serum depends on T and B cell stimulating protein B. Infect Immun 83:1257-1264, which is hereby incorporated by reference in its entirety.

[0104] Table 8 lists the components of the chemically defined medium at 50 times and 100 times concentrated stock solutions. All solutions are made in deionized water and filtered through a sterile 0.22 micron filter. The amino acid solutions were adjusted to pH 7 -7.5. Iron citrate, iron EDTA, iron silicate, iron pyridone, and iron pyrophosphate solutions were prepared by heating the mixture of water and iron complex to 50 °C with agitation. TheAttorney Docket No. OMV0005-401-PCiron solutions (pH ~ 3) were prepared fresh, weekly. Cysteine and cystine stock solutions were prepared freshly in 0.4M hydrochloric acid. All the components were combined with the iron solution followed by the cystine solution added last. The ferric iron concentration for all iron supplements except for human transferrin was 8 mM. The final concentration of recombinant human transferrin (Optifemn, InVitra, St. Louis MO) tested was 0.75, 1, 1.25, 1.5, 1.75, and 2 grams per liter (Figure 2). Recombinant lactoferrin and hemoglobin were tested individually at 1 g / L and 2 g / L. The pH of the medium was adjusted to 7.4, the volume was adjusted to lx of stock solutions with deionized water and the medium was filtered using a sterile 0.22 micron filter.Table 8.50x stock solutions lOOx stock solutionsChemical Amount (g / L) Chemical Amount (g / mL) L-glutamic acid 195 Potassium sulfate 100L-arginine 7.5 Ammonium chloride 40L-glycine 12.5 Magnesium chloride 40L-serine 25 Calcium chloride 3Sodium chloride 290Potassium phosphate 200(mono basic)Glucose 500D, L lactate 18Cysteine 0.4hydrochlorideCystine 1.8Example 4

[0105] This example compares the growth of Neisseria meningitidis strain H44 / 76 in chemically defined medium containing different ferric iron supplements.

[0106] The bacteria were streaked on chocolate agar plates (Remel, Thermo Fisher Scientific, Pittsburgh, PA) and grown overnight at 37 °C in an atmosphere of ambient air supplemented with 5% CO2. Individual colonies were isolated from the chocolate agar plates using a bacterial loop and dispersed in 4 individual tubes containing 5 milliliters of medium such that the OD620nm (Spectronic 200, Thermo Fisher Scientific) was between 0.15 and 0.2. The bacterial cultures were grown at 37 °C until the OD620nm was approximately 0.6. The cultures were then added to 90 milliliters of fresh medium warmed to 37 °C in a shake flask. The flasks were shaken at 200 rpm in an incubator at 37 °C, 5% CO2 for the indicated time. The optical density at 620nm was recorded at 1-hour intervals.Attorney Docket No. OMV0005-401-PC

[0107] Figure 1 shows that optical density at 620nm of Neisseria meningitidis strain H44 / 76 cultures in chemically defined medium with ferric iron supplements reaches a maximum of approximately 5 or less than after 8 hours of growth. In contrast, bacteria grown in the same medium supplemented with human transferrin produced in rice continues to grow for 12 hours reaching an OD620nm of 15.

[0108] Figure 2 shows that the highest bacterial culture densities were achieved when supplementing the chemically defined medium with >1.25 grams of transferrin per liter of medium and batch feeding with all components of the medium including iron citrate to regenerate holo transferrin.

[0109] Neisseria meningitidis can also acquire iron from human lactoferrin. However, as shown in Figure 3, chemically defined medium supplemented with 2 grams per liter of human lactoferrin produced in rice (InVitra, St. Louis, MO) does not support growth of the bacteria to the high densities achieved with 2 grams / liter of human transferrin.

[0110] The results show that recombinant human transferrin produced in rice is effective in sustaining growth of Neisseria meningitidis to the high densities needed for commercial production of NOMV. Naturally blebbed vesicles that are of interest for vaccines are produced by other bacterial species. Recombinant transferrin may be also support growth of bacteria that acquire iron through transferrin receptors (Table 11) to similarly high densities that allow for the production of NOMV sufficient for a commercial product.Example 5

[0111] This example shows that the production of Neisseria meningitidis in a large fermenter is greatly improved with the use of chemically defined medium containing human transferrin.

[0112] Bacterial cultures were seeded as described in Example 4 using chemically defined medium described in Example 3, except for transferrin being 1.5 grams / liter, to produce an inoculum culture for a 30L fermenter. At each seed stage, the bacteria were transferred to a larger volume once the OD620nm reached 0.6 such that when the culture was diluted in the larger volume the OD620nm in the larger volume was approximately 0.15-0.25. This process was repeated until 3L of bacterial culture having an OD620nm of approximately 1.5-2.0 was produced. The 3L of bacterial culture was then transferred into 20L of chemically defined medium containing 1.5 grams per liter of human transferrin (Optiferrin, InVitra, St. Louis, MO). The growth curve of the 30- liter fermenter is shown in Figure 4.Attorney Docket No. OMV0005-401-PC

[0113] Provided below are sequences of transferrin proteins. In some embodiments, the transferrin disclosed herein may be a variant of human transferrin. In some embodiments, the variant of human transferrin may include substitutions in domains other than the iron binding domain, or in residues that are not involved in iron binding. In some embodiments, the variant of human transferrin may include substitutions in domains or residues that are not conserved or are less conserved amongst transferrin sequences in other species. Recombinant transferrin proteins are disclosed in, e.g., US9321828 (issuing from US20120088729A1. Ventria Bioscience Inc.), US20140031294A1 (Novozymes Biopharma DK AS), and US20060205037 (Biorexis Pharmaceutical Corp.).Table 9.Identifier Protein Sequence MRLAVGALLVCAVLGLCLAVPDKTVRWCAVSEHEA TKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRA IAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYG SKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTG LGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSG SCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGA FKCLKDGAGDVAFVKHSTIFENLANKADRDQYELL CLDNTRKPVDEYKDCHLAQVPSHTWARSMGGKEDmature LIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFK human TF DSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTC SEQ ID NO: 49Swiss-Prot PEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKI P02787 ECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGK CGLVPVLAENYNKSDNCEDTPEAGYFAVAWKKSA SDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKI NHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLC EPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQN TGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYAN CHLARAPNHAWTRKDKEACVHKILRQQQHLFGSN VTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNT YEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP MRLAVGALLVCAVLGLCLAVPDKTVRWCAVSEHEA TKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRA IAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYG SKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGSerotransf LGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGerrin | HomoSEQ ID NO: 50 SCAP CADGTDFPQLCQLCPGCGCSTLNQYFGYSGA sapiens N FKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLlobeCLDNTRKPVDEYKDCHLAQVPSHTWARSMGGKED LIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFK DSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTC PEAPTSerotransf DECKPVKWCALSHHERLKCDEWSVNSVGKIECVSA SEQ ID NO: 51errin | Homo ETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPAttorney Docket No. GMV0005-401-PCsapiens C VLAENYNKSDNCEDTPEAGYFAVAWKKSASDLTW lobe DNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRF DEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNK EGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKN PDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLAR APNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCS GNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRPExample 6

[0114] This example describes the preparation of a medium that supports the growth of Neisseria gonorrhoeae bacteria to high densities needed for the production of native outer membrane vesicles (NOMV) that can be used as vaccines to elicit antibodies to antigens providing protection against disease.

[0115] Table 10 lists the components of the supplements added to the Gonococcal base liquid medium (GCBL) to make concentrated stock solutions. The supplements are made in deionized water and sterilized by filtration through a 0.22 micron filter. Modified Kellogg’s Supplement 1 was prepared fresh, weekly. Kellogg’s Supplement 2 was prepared and stored at -20°C for not longer than a week. For the preparation of the complete GCBL. 15 g of Proteose Peptone #3, or Vegie Peptone™ from Millipore Sigma, 4 g of K2HPO4, 1 g of KH2PO4, and 1 g of NaCl are dissolved in 0.8 L of deionized water. 10 ml of Modified Kellogg’s Supplement 1, and 1 ml of Kellogg's Supplement 2 are then added. Recombinant human transferrin (Optiferrin, InVitra, St. Louis MO) was added to GCBL at 2 grams per liter. The pH of the medium was adjusted to 7.2, the volume was adjusted to 1 L solution with deionized water and the medium sterilized by filtration using a 0.22 micron filter.Table 10.lOOx Modified lOOOx Kellogg’sKellogg’s Supplement 2Supplement 1Chemical Amount (g / L) Chemical Amount (g / mL) Glucose 40 Ferric Nitrate 0.0005 Glutamine 1Thiamine 0.002pyrophosphateSodium bicarbonate 42Attorney Docket No. OMV0005-401-PCExample 7

[0116] This example shows the growth of Neisseria gonorrhoeae strain FA1090, WHO F, WHO M and WHO N mutants, where LpxLl and rmpM genes were knocked out, in GCLB medium containing recombinant human transferrin. Also, the strains were grown in the GCLB medium with either Peptone #3 from an animal source or Veggie™ Peptone derived from a vegetable source as described above.

[0117] The bacteria were streaked on Chocolate II Agar plates (Remel, Thermo Fisher Scientific, Pittsburgh, PA) and grown overnight at 37 °C in an atmosphere of ambient air supplemented with 5% CO2. Individual colonies were isolated from the chocolate agar plates using a bacterial loop and re-streaked on a new Chocolate II Agar plate. Bacteria were then grown overnight at 37 °C in an atmosphere of ambient air supplemented with 5% CO2. After this second growth on an agar plate, individual colonies were dispersed in 100 ml of liquid medium such that the OD620mn (Spectronic 200, Thermo Fisher Scientific) was between 0.15 and 0.2 in a shake flask. The flask was shaken at 200 rpm in an incubator at 37 °C, 5% CO2 for the indicated time. The optical density at 620nm was recorded at different timepoints.

[0118] The optical density at OD620nm of Neisseria gonorrhoeae strain FA1090 mutant cultures in liquid medium supplemented with human transferrin produced in rice reached an OD620nm of 7.6 when animal derived Peptone #3 was used or 8 when using Peptone derived from a plant source (Veggie™ Peptone, MilliporeSigma) after 23 hours of incubation. The result shows that recombinant human transferrin produced in rice is effective in sustaining growth of Neisseria gonorrhoeae to the high densities needed for commercial production of NOMV.Example 8

[0119] This example shows the purification of NOMV using size exclusion chromatography (SEC). Bacteria from Example 5 were collected by centrifugation. The supernatant containing the NOMV was concentrated by tangential flow filtration (TFF) using a lOOkD membrane (Pall, Washington, NY), and diafiltered into a buffer containing 10 mM Tris, pH 8, and 2mM magnesium chloride (Pall, Washington, NY). SEC was performed using a Sephacryl S-400 column (Cytiva, Marlborough, MA). An example of the SEC cycle is shown in Figure 5. Peak 1 represents the purified NOMV as shown in an SDS-PAGE result in Figure 6 (line 8, 9 and 10). Transferrin and other impurities are found in Peak 2 through Peak 6.Attorney Docket No. OMV0005-401-PC

[0120] The data shows that Neisseria meningitidis bacteria cultured in chemically defined medium supplemented with holo human transferrin results in >3 -fold higher densities of bacteria and up to 10-fold higher production of NOMV (Table 12).Table 11. Pathogens that acquire ferric iron through transferrin-specific receptor proteins.Host Pathogen Disease Lineage Human Neisseria meningitidis Meningitis, sepsis Neisseriaceae Neisseria gonorrhoeae Gonorrhea Neisseriaceae Haemophilus Meningitis, sepsis, pneumonia Pasteurellaceae influenzae(type b)H. influenzae Otitis media, chronic obstructive Pasteurellaceae (non-typeable) pulmonary disease (COPD),pneumoniaMoraxella catarrhalis Otitis media, COPD Moraxcllaccae Cattle Mannheinria Bovine respiratory disease Pasteurellaceae haemolytica (BRD)Pasteurella multoeida Hemorrhagic septicemia (HS), Pasteurellaceae BRDH istophilus somni BRD, thrombotic Pasteurellaceae meningoencephalitis (TME),myocarditisMoraxella bovis Pinkeye (infectious bovine Moraxellaceae keratoconjuncti vitis) IB KSheep Bibersteinia trehalosi Septicemia, pneumonia Pasteurellaceae Pig Actinobacillus Pleuropneumonia Pasteurellaceae pleuropneunioniaeHaemophilus parasuis Glasser’s disease Pasteurellaceae 4 ctinobacill us suis Pneumoni a, septicemi a Pasteurellaceae Poultry Avibacterium sp. None PasteurellaceaeAttorney Docket No. GMV0005-401-PCTable 11. NOMV yieldCulture Volume Ferric Iron SupplementFerric Citrate Transferrin0.1 L Img / L 12mg / L1 L 1.5mg / L lOmg / L30 L N / A lOOmg / LExample 9

[0121] Immunogenicity of NOMV from meningococcal and gonococcal production strains in GDI mice.

[0122] This example shows the IgG titers of the MenB NOMV-FHbp (i.e. NOMV-FHbp ID9 mutant and NOMV-FHbp ID22 mutant) and Ng NOMV-Ng (i.e. NOMV-FA1090 and NOMV-WHO F) bi-valent components and the combination NOMV-combo vaccine determined in groups (N=10 mice per group) of four- to six-week-old outbred CD-I mice. Each dose contains a total of 2.5 pg of individual NOMV from NOMV-FHbp ID9 and NOMV-FHbp ID22 combined in a ratio of 1:1, respectively, 5pg for each bi-valent NOMV-FHbp and NOMV-Ng when combined (i.e. lOpg total in NOMV-combo). The vaccines were adsorbed to 100 pg of aluminum hydroxide (Alhydrogel) in lOmM Tris, pH 7.5 buffer containing 0.9% (weight / volume) sodium chloride and 3% (weight / volume) sucrose that provides long-term stability to the NOMV vaccine. Control groups of mice received the licensed 4CMenB vaccine (1 / 5 of a human dose, which contains 5 pg of dOMV), or aluminum adjuvant in the same buffer solution as the other vaccines. Two doses of the vaccines were given i.p. 3 weeks apart and serum samples obtained after 1 dose and 2 doses 15 days after each dose to measure increases in antibody titers after each dose.ELISA assay

[0123] To determine IgG titers for FHbp ID22 and ID9 and PorB.lA and PorB.lB, 96-well plates (Nunc) were coated overnight at 4°C with recombinant 1D22 and ID13 (surrogate for ID9) or NOMV from Ng strains FA1090 (PorB.lB) or WHO F (PorB.lA). Plates were blocked with 1% BSA + 0.05% Tween 20 in PBS. The mouse sera were diluted in PBS + 0.1 % Tween 20 and added to plates for 2 hours. Plates were stained with alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson Immuno Research Laboratories) (1:2,000) or goat anti-rabbit IgG (Jackson Immuno Research Laboratories) (1:2,000) for 1 hour and developed using p-nitrophenyl phosphate (Thermo Fisher Scientific). Results for binding to recombinantAttorney Docket No. OMV0005-401-PCFHbp ID22 and ID13 and NOMV from FA 1090 and WHO F Ng strains were determined by ELISA are summarized in Table 13, below.

[0124] The results show that each component was able to elicit antibodies specific for the vaccine antigen present in each component as an NOMV-FHbp or NOMV-Ng bi-valent vaccine or when combined as a tetra-valent combination vaccine (NOMV-combo).Table 13. IgG titers determined by ELISA.Antigen Antiserum tested GMT (±95% CI)NOMV-FHbp NOMV-Ng NOMV-combo 4C-MenB rFHbp ID 13 67,940 Below LLOD* 20,620 Not tested (18,460 / 115,220) (9,910 / 42,900) rFHbp ID22 105,880 Below LLOD* 27,710 Not tested (37,110 / 161,120) (10,930 / 70,290) Ng strain 7,687 206,710 144,620 Not TestedFA 1090 (3,070 / 19,230) (102,960 / 415,010) (77,840 / 268,670)NOMVNg strain 10,137 248,790 141891 Not tested WHO F (4,380 / 23,470) (142,370 / 434.760) (42,700 / 471,500)NOMV*LLOD, lower limit of detection.Example 10

[0125] The serum bactericidal activity (SBA) is a surrogate for protection against disease caused by Neisseria meningitidis. Although SBA has not been established as a surrogate for protection against N. gonorrhoeae, SBA shows that there is sufficient antigen on the surface of bacteria to activate mechanisms of immune clearance. An established SBA assay (Del Tordello et al. 2012; Beernink 2018) was used to measure the ability of serum antibodies to kill Nm and Ng in the presence of human complement performed similarly to that recently described (Zhu et al. 2024). The test strains were grown in the presence of CMP-NANA, which results in sialylation of LOS as occurs in the human host. For these assays, a commercial source of complement (20%) pooled from human donors that has been depleted of IgG and IgM antibodies (PeLFreez, Rodgers, AR) to remove any naturally-occurring antibodies and validated for hemolytic complement activity (Bccmink 2018). The SBA titer is the interpolated serum dilution that results in 50% survival of the bacteria compared to negative control wells. The results of SBA assay for test strains representing FHbps from subfamilies A and B and Ng PorB subfamilies 1 A and IB are summarized in Table 14, below.

[0126] The results show that each bi-valent NOMV-FHbp and NOMV-Ng vaccine elicited SBA against diverse MenB and Ng strains, respectively, and against both diverseAttorney Docket No. OMV0005-401-PCmeningococcal and gonococcal strains tested when given as a tetra-valent combination vaccine (NOMV-combo).Table 14. SBA titers of antibodies elicited by vaccination with two doses of the NOMV vaccines or the MenB vaccine MenB-4C (Bexsero®).MenB strains (ID, Antiserum tested (GMT ±95% CI)subfamily)NOMV- NOMV-Ng NOMV-combo 4C-MenB FHbp240648 (24, A) 610 Below LLOD* 180 Below LLOD*(50 / 7,370) (15 / 2,050)M01239 (28, A) 3,720 Below LLOD* 50 Below LLOD*(13.2 / 10,520) (3 / 880)240476 (13, B) 128 Below LLOD* 110 Not tested (10 / 1,670) (3.1 / 3,980)Quebec (15, B) 560 Below LLOD* 57 180(51 / 6,180) (17 / 190) (1 / 500) Ng strains (PorB Antiserum testec (GMT ±95% CI)subfamily)FA1090 (IB) Below LLOD 6,480 3,570 Below LLOD*(3,780 / 11,100) (1,880 / 6,790) WHO F (IA) 58 3,430 1,180 Not tested (32 / 105) (1,250 / 9,400) (100 / 13,500) WHO G (IB) Below LLOD 2,872 1,083 Not tested (690 / 12,000) (310 / 3,740)WHO M (IB) Below LLOD 3,427 2,550 Not tested (1,400 / 8,500) (450 / 14,600)WHO N (IA) Below LLOD 450 305.1 Not tested(64 / 3,150) (29 / 3,150)*LLOD Lower limit of detectionExample 11.

[0127] The objective of this study was to assess pyrogenicity of NOMV-FHbp compared with MenB-4C in the standard Rabbit Pyrogen Test. NOMV from wild-type N. meningitidis bacteria contain lipooligosaccharide (LOS) endotoxin activity that is not acceptable for use in humans. The NOMV used in the OMVax bi-valent NOMV-FHbp vaccine was produced from a MenB strain that has been genetically modified by deletion of the IpxlLl gene resulting in the production of LOS that is penta-acylated instead of hexaacylated (Steeghs et al. 2004). The presence of only penta-acyl LOS in the parent to the MCB strains was confirmed by ESI-MS analysis of a preparation of LOS from the parent strain by the Biotechnology Center at the University of Wisconsin, Madison. Penta-acyl LOS has beenAttorney Docket No. GMV0005-401-PCshown to have atenuated endotoxin activity that is equal to or less than detergent extracted OMV made from wild- type N. meningitidis and has been used in OMV-based vaccines in several countries including Cuba, Norway, South America and New Zealand over many years to stem meningococcal outbreaks. dOMV is also a component of the 4CMenB vaccine approved for use in several countries in individuals as young as infants and in all other age groups.

[0128] The study was performed by Charles River Laboratories, Ballina, Ireland using adult New Zealand White rabbits larger than 1.8 kg. Groups of 3 rabbits were given NOMV-FHbp absorbed to Rehydragel LV® or 4CMenB at the proposed human dose 0.5mcg / kg. The rabbits were clinically examined immediately prior to each pyrogen test. Temperatures of the animals were recorded, beginning at least 90 minutes at 30-minute intervals before injection. Post injection temperature was recorded at Vi, 1, I G, 2, 21 / 2, and 3 hours subsequent to the injection. A second study compared NOMV-FHbp without and with absorbtion to Alhydrogel®.

[0129] In addition to the temperature monitoring (by rectal probe using a thermocouple reading device and thermocouples) the health status of the animal was visually assessed throughout the test for any abnormal physical reactions to the test sample injection. The test material was injected by a slow intravenous injection in the marginal ear of the rabbit. Subsequently, 6 visual assessments and temperature recordings on the animal were made at 30-minute intervals, until the end of the test. The test was performed under license from the Health Products Regulatory Authority (HPRA - Licence No. AE19108 / P074) and the animal welfare considerations were assessed and approved by the Ethics Committee at Charles River Laboratories Ireland Ltd. and found to be acceptable.

[0130] Agents that pass the Rabbit Pyrogen Test will have no abnormal physical reactions and have a total temperature rise of <0.5°C. Rabbits given NOMV-FHbp vaccine had a total temperature rise for the 3 of 0.2°C compared with 0.4°C for rabbits given MenB-4C. Rabbits given NOMV-FHbp either without or with absorption to Alhydrogel® had a total temperature rise for the 3 rabbits of 0.3.Example 12.

[0131] To measure inhibition of colonization, Ng test strains grown in the presence of CMP-NANA, were labeled using the CellTrace™ CFSE Cell Proliferation Kit (ThermoFisher). Human ME- 180 cervical and VK2 vaginal cells were cultured in the wells ofAttorney Docket No. OMV0005-401-PCa 24 well plate in the appropriate culture medium. IgG was purified from pooled mouse serum from each vaccine group using Protein G. IgG purification is necessary because nonspecific IgM antibodies present in control serum can inhibit binding of Ng to ME-180 cells. The purified IgG at 1:25 dilution was combined with the target cells. After a 4 hr incubation at 37°C in 5% CO2, the unbound bacteria were removed by multiple washes and the cells are labeled with PerCP-conjugated anti-CD47 antibody after cells are released from the culture plates with Accutase (ThermoFisher). The cells and bound bacteria are then analyzed by ImageStream (Agilent) that combines flow cytometry with fluorescence microscopy. The ImageStream analysis allows for determining the number of cervical or vaginal cells with bacteria bound to them. As described in statistical methods below, N=4 wells per test point. An example of inhibition of colonization of several diverse Ng strains representing subfamilies expressing either PorB.lA or PorB.lB by purified IgG from serum of mice immunized with the NOMV-FHbp, NOMV-Ng and NOMV-combo vaccines

[0132] A vaccine that can elicit antibodies that inhibit colonization arc especially important in preventing gonorrhea. As shown in Table 15, purified IgG from mice immunized with bi-valent NOMV-FHbp or NOMV-Ng or the combined tetravalent NOMV-combo vaccines were all able to inhibit colonization of diverse Ng strains compared to bacteria alone or control IgG isolated from the serum of mice immunized with alum adjuvant alone. The NOMV-Ng and NOMV-combo vaccines were the most effective.Table 15. Inhibition of adhesion to human ME-180 cervical cells by purified IgG from mice vaccinated with 2 doses NOMV vaccines.Purified IgG testedMean percent inhibition(±SD)Ngo strains (PorB NOMV-FHbp NOMV-Ng NOMV-combosub-family)FA1090 (IB) 2.0 10.0 5.4(0.7 / 3) (0.795 / 18.4) (4.3Z6.5)WHO F (1A) 1.6 50.9 33.3(1.2 / 2.1) (43.8 / 58) (26 / 40.7)WHO G (IB) 22.6 26.0 28.7(16.3 / 29) (25.38 / 26.58) (27.4 / 30)WHO M (IB) 8 11.64 2.4(6.5 / 9.3) (9.2 / 14.1) (0.89 / 4.8)WHO N (1A) 14.9 22.72 32.4(5.6 / 24.2) (16.6 / 28.8) (22.4 / 42.4) MS 11 (IB) 23.2 43.7 37.3(12.4 / 34) (42.8 / 44.7) (36.1 / 38.5)Calculated as ((CFU adjuvant only control-CFU test IgG) / (CFU adjuvant only control))xl00.Attorney Docket No. OMV0005-401-PC

[0133] The functional ability of individual and combination vaccine antisera to inhibit colonization of Ng will be evaluated in a human CEACAM1 / FH transgenic mouse model of Ng colonization. Since the 1930s, a wide range of animals have been tested as models of meningococcal and gonococcal disease including monkeys, rabbits, guinea pigs, adult and infant rats and mice and chicken embryos (Arko 1989; Jerse et al. 2011). Two factors unique to humans appear to be essential for an animal model that is both colonized by Nm and results in bacteremia and / or meningitis or, for Ng, genital tract infection. The first is a mechanism of attachment (Johansson et al. 2003; Johswich et al. 2013) and the second is a mechanism of immune shielding ((Gill and Atkinson 2004) and as described below). Nutrient acquisition is also important, particularly iron (Arko 1989), but Neisseria have the capability to scavenge iron from sources in the mouse. Also, proteins such as human holo transferrin or lactoferrin can be provided exogenously (Yi, Stephens, and Stojiljkovic 2003).ENUMERATED EMBODIMENTS

[0134] The disclosure provides also the following non- limiting embodiments.

[0135] Embodiment 1. A process for producing outer membrane vesicles (OMVs) from bacteria that acquire iron from human transferrin comprising growing the bacteria in medium comprising recombinant human transferrin.

[0136] Embodiment 2. The process of embodiment 1, wherein the process comprises:a) growing the bacteria in an atmosphere of ambient air supplemented with 3 % - 5 % CO2;b) isolating individual cultures of bacteria and dispersing the cultures in medium comprising recombinant human transferrin;c) growing the redispersed cultures at about 37 °C;d) adding fresh medium and further growing the cultures at about 37 °C in an atmosphere of ambient air supplemented with CO2 one or more times; and e) purifying the cultures.

[0137] Embodiment 3. The process of either of embodiments 1 and 2, wherein the medium contains negligible detergent or is detergent free.

[0138] Embodiment 4. The process of any of embodiments 1-3, wherein the OMVs are free of EDTA.Attorney Docket No. OMV0005-401-PC

[0139] Embodiment 5. The process of any of embodiments 1-4, wherein the cultures are purified by size exclusion chromatography.

[0140] Embodiment 6. The process of any of embodiments 1-5, wherein recombinant transferrin is human.

[0141] Embodiment 7. The process of any of embodiments 1-6, wherein the recombinant human transferrin has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:49.

[0142] Embodiment 8. The process of any of embodiments 1 -7, wherein the recombinant human transferrin comprises a C-lobe which has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:51.

[0143] Embodiment 9. The process of any of embodiments 1-8, wherein the recombinant transferrin is produced in a plant.

[0144] Embodiment 10. The process of embodiment 9, wherein the plant is a monocot plant.

[0145] Embodiment 11. The process of embodiment 10, wherein the plant is a plant from the family Gramineae.

[0146] Embodiment 12. The process of embodiment 11, wherein the plant is a rice plant (Oryza sps.).

[0147] Embodiment 13. The process of any of embodiments 1-12, wherein the recombinant transferrin is produced in rice.

[0148] Embodiment 14. The process of any of embodiments 1-13, wherein the bacteria is chosen from human pathogenic Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae (type B), H. influenzae (non-typeable), Moraxella catarrhalis, extraintestinal pathogenic Esherichia coli, and Salmonella typhimurium SL1344.

[0149] Embodiment 15. The process of embodiment 14, wherein the bacteria is chosen from human pathogenic Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae (type B), and H. influenzae (non-typeable).

[0150] Embodiment 16. The process of embodiment 15, wherein the bacteria is human pathogenic Neisseria meningitidis.

[0151] Embodiment 17. The process of embodiment 16, wherein the human pathogenic Neisseria meningitidis does not express one or more, or two or more, or three or more, or four or more, of porin protein PorA, hexa-acyl lipooligosaccharide (LOS), capsular polysaccharide, and sialylated LOS.Attorney Docket No. GMV0005-401-PC

[0152] Embodiment 18. The process of embodiment 17, wherein the human pathogenic Neisseria meningitidis does not express the porin protein PorA.

[0153] Embodiment 19. The process of embodiment 17, wherein the human pathogenic Neisseria meningitidis does not express hexa-acyl lipooligosaccharide (LOS).

[0154] Embodiment 20. The process of embodiment 17, wherein the human pathogenic Neisseria meningitidis does not express capsular polysaccharide.

[0155] Embodiment 21. The process of embodiment 17, wherein the human pathogenic Neisseria meningitidis produces penta-acyl lipooligosaccharide (LOS).

[0156] Embodiment 22. The process of embodiment 17, wherein the human pathogenic Neisseria meningitidis has a knocked out gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.

[0157] Embodiment 23. The process of embodiment 17, wherein the human pathogenic Neisseria meningitidis has a replacement of wild type (WT) Factor H binding protein (FHbp) subfamily B, siaD-galE, and IpxLl genes with subfamily A FHbp ID22 mutant comprises one or more of L130R, G133D, and K218N substitutions.[001581 Embodiment 24. The process of embodiment 23, wherein the subfamily A FHbp ID22 mutant comprises two or more of L130R, G133D, and K218N substitutions.

[0159] Embodiment 25. The process of any one of embodiments 23-24, wherein the subfamily A FHbp ID22 mutant comprises three of L130R, G133D, and K218N substitutions.

[0160] Embodiment 26. The process of embodiment 17, wherein the human pathogenic Neisseria meningitidis has a replacement of WT FHbp subfamily B, siaD-galE, and IpxLl genes with subfamily B FHbp ID9 single mutant with S228R substitution.

[0161] Embodiment 27. The process of embodiment 22, wherein the human pathogenic Neisseria meningitidis has a knocked out or disrupted IpxLl gene.

[0162] Embodiment 28. The process of embodiment 22, wherein the human pathogenic Neisseria meningitidis has knocked out or disrupted siaD and GalE genes.

[0163] Embodiment 29. The process of embodiment 22, wherein the human pathogenic Neisseria meningitidis has a knocked out or disrupted FHbp gene.

[0164] Embodiment 30. The process of embodiment 16, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of a gene product from a gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.Attorney Docket No. GMV0005-401-PC

[0165] Embodiment 31. The process of embodiment 30, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of IpxLl.

[0166] Embodiment 32. The process of embodiment 30, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of siaD and GalE.

[0167] Embodiment 33. The process of embodiment 30, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of FHbp.

[0168] Embodiment 34. The process of embodiment 30, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of a gene product from a gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.

[0169] Embodiment 35. The process of embodiment 16, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of IpxLl.

[0170] Embodiment 36. The process of embodiment 35, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of siaD and GalE.

[0171] Embodiment 37. The process of embodiment 35, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of FHbp.

[0172] Embodiment 38. The process of embodiment 16, wherein the human pathogenic Neisseria meningitidis does not express sialylated LOS.

[0173] Embodiment 39. The process of embodiment 16, wherein the human pathogenic Neisseria meningitidis has a reduced ability to bind human Factor H.

[0174] Embodiment 40. The process of embodiment 39, wherein the human pathogenic Neisseria meningitidis has a reduced ability to bind human Factor H due to a replacement of the factor II binding protein gene with DNA coding for a Factor II binding protein gene expressing a mutant Factor H binding protein with a reduced ability to bind human Factor H.

[0175] Embodiment 41. The process of either of any of embodiments 16 to 40, wherein the human pathogenic Neisseria meningitidis is transformed with a multicopy plasmid that contains an engineered promoter for overexpressing a mutant Factor H binding protein with reduced Factor H binding.

[0176] Embodiment 42. The process of embodiment 16, wherein the human pathogenic Neisseria meningitidis has at least two of, or at least three of, or at least four of, or at least five of, or all of:a) does not express porin protein PorA;b) does not express capsular polysaccharide;Attorney Docket No. GMV0005-401-PCc) does not express sialylated LOS;d) produces penta-acyl lipooligosaccharide instead of hexa-acyl LOS; e) has a replacement of the factor H binding protein gene with DNA coding for a Factor H binding protein gene expressing a mutant Factor H binding protein with reduced ability to bind human Factor H; andf) is transformed with a multicopy plasmid that contains an engineered promoter for overexpressing a mutant Factor H binding protein with reduced Factor H binding.

[0177] Embodiment 43. The process of any of embodiments 16 to 42, wherein the human pathogenic Neisseria meningitidis is H44 / 76.

[0178] Embodiment 44. The process of any of embodiments 1 to 43, wherein the human pathogenic Neisseria meningitidis bacteria cultured in medium comprising human transferrin results in at least 3-fold higher densities of bacteria.

[0179] Embodiment 45. The process of embodiment 44, wherein the human pathogenic Neisseria meningitidis bacteria cultured in medium comprising human transferrin results in at least 5 -fold higher production of OMVs.

[0180] Embodiment 46. The process of any of embodiments 1 to 45, wherein the human pathogenic Neisseria meningitidis bacteria cultured in medium comprising human transferrin is grown for about 6 to about 16 hours.

[0181] Embodiment 47. The process of embodiment 46, wherein the human pathogenic Neisseria meningitidis bacteria cultured in medium comprising human transferrin is grown for about 10 to about 14 hours.

[0182] Embodiment 48. The process of embodiment 47, wherein the human pathogenic Neisseria meningitidis bacteria cultured in chemically defined medium comprising human transferrin is grown for about 12 hours.

[0183] Embodiment 49. The process of any of embodiments 1 to 48, wherein the medium is chosen from Catlin 6 medium, gonococcal (GC) medium, Frantz medium, modified Frantz medium, Muller-Hinton medium, Thayer-Martin agar, modified Thayer-Martin agar, New York City (NYC) agar, chocolate agar, and GC agar base.

[0184] Embodiment 50. The process of embodiment 49, wherein the medium is Catlin 6 medium.

[0185] Embodiment 51. The process of embodiment 49, wherein the chemically defined culture medium is supplemented with glucose, D, L-lactate, cysteine, cystine, or combinations thereof.Attorney Docket No. OMV0005-401-PC

[0186] Embodiment 52. The process of any of embodiments 1 to 51, wherein the chemically defined culture medium is supplemented with recombinant human transferrin.

[0187] Embodiment 53. The process of any of embodiments 1 to 52, wherein the chemically defined culture medium comprises between about 0.5 and about 5 g / L recombinant human transferrin.

[0188] Embodiment 54. The process of embodiment 53, wherein the chemically defined culture medium comprises between about 0.75 and about 2 g / L recombinant human transferrin.

[0189] Embodiment 55. The process of embodiment 54, wherein the chemically defined culture medium comprises between about 1.25 and about 2 g / L recombinant human transferrin.

[0190] Embodiment 56. The process of any of embodiments 1 to 55, wherein the chemically defined culture medium comprises about 0.75, about 1, about 1.25, about 1.5, about 1.75, or about 2 g / L recombinant human transferrin.

[0191] Embodiment 57. The process of embodiment 56, wherein the chemically defined culture medium comprises about 1.5 g / L recombinant human transferrin.

[0192] Embodiment 58. The process of embodiment 15, wherein the bacteria is human pathogenic Neisseria gonorrhoeae.

[0193] Embodiment 59. The process of embodiment 58, wherein the human pathogenic Neisseria gonorrhoeae has decreased or knocked-out expression and / or function of the lipid A biosynthesis lauroyl acyltransferase (IpxLl) gene and reduction of expression of the modifiable protein (rmp) gene.

[0194] Embodiment 60. The process of embodiment 59, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out gene selected from one or more, or two or more, of IpxLl and rmp.

[0195] Embodiment 61. The process of embodiment 60, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out or disrupted IpxLl gene.

[0196] Embodiment 62. The process of embodiment 61, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out or disrupted rmp gene.

[0197] Embodiment 63. The process of embodiment 58, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of a gene product from a gene selected from one or more, or two or more, of IpxLl and rmp.

[0198] Embodiment 64. The process of embodiment 63, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of IpxLl.Attorney Docket No. GMV0005-401-PC

[0199] Embodiment 65. The process of embodiment 63, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of rmp.

[0200] Embodiment 66. The process of embodiment 58, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of a gene product from a gene selected from one or more, or two or more, of IpxLl and rmp.

[0201] Embodiment 67. The process of embodiment 66, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of IpxLl.

[0202] Embodiment 68. The process of embodiment 66, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of rmp.

[0203] Embodiment 69. The process of any of embodiments 58-68, wherein the human pathogenic Neisseria gonorrhoeae strain is chosen from FA1090, WHO F, WHO G, WHO N, WHO M, and MSI 1.

[0204] Embodiment 70. The process of any of embodiments 58-69, wherein the chemically defined culture medium is GCLB medium.

[0205] Embodiment 71. The process of embodiment 70, wherein the chemically defined culture medium is supplemented with Peptone #3 from an animal source or Veggie™ Peptone derived from a vegetable source, or combinations thereof.

[0206] Embodiment 72. The process of any of embodiments 58-71, wherein the chemically defined culture medium is supplemented with recombinant human transferrin.

[0207] Embodiment 73. The process of any of embodiments 58-72, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin results in at least 3-fold higher densities of bacteria.

[0208] Embodiment 74. The process of embodiment 73, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin results in at least 5-fold higher production of OMVs.

[0209] Embodiment 75. The process of any of embodiments 58-74, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 6 to about 16 hours.

[0210] Embodiment 76. The process of embodiment 75, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 10 to about 14 hours.Attorney Docket No. OMV0005-401-PC

[0211] Embodiment 77. The process of embodiment 76, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 12 hours.

[0212] Embodiment 78. The process of any of embodiments 58-77, wherein the culture medium comprises between about 0.5 and about 5 g / L recombinant human transferrin.

[0213] Embodiment 79. The process of embodiment 78, wherein the culture medium comprises between about 0.75 and about 2 g / L recombinant human transferrin.

[0214] Embodiment 80. The process of embodiment 79, wherein the culture medium comprises between about 1.25 and about 2 g / L recombinant human transferrin.

[0215] Embodiment 81. The process of any of embodiments 58-80, wherein the culture medium comprises about 0.75, about 1, about 1.25, about 1.5, about 1.75, or about 2 g / L recombinant human transferrin.

[0216] Embodiment 82. The process of embodiment 81, wherein the culture medium comprises about 1.5 g / L recombinant human transferrin.

[0217] Embodiment 83. The process of any of embodiments 1-82, wherein the OMVs are native OMVs (NOMVs) blebbed from mutant strains of Neisseria.

[0218] Embodiment 84. The process of any of embodiments 1-82, wherein the OMVs are produced by genetic, chemical or mechanical methods from mutant strains of Neisseria.

[0219] Embodiment 85. A composition comprising outer membrane vesicles (OMVs), bacteria that acquire iron from human transferrin, and recombinant human transferrin.

[0220] Embodiment 86. The composition of embodiment 85, wherein the medium contains negligible detergent or is detergent free.

[0221] Embodiment 87. The composition of embodiment 85, wherein the OMVs are free of EDTA.

[0222] Embodiment 88. The composition of any of embodiments 85-87, wherein the recombinant human transferrin has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:49.

[0223] Embodiment 89. The composition of any of embodiments 85-88, wherein the recombinant human transferrin comprises a C-lobe which has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:51.

[0224] Embodiment 90. The composition of any of embodiments 85-89, wherein the recombinant human transferrin is produced in a plant.Attorney Docket No. GMV0005-401-PC

[0225] Embodiment 91. The composition of embodiment 90, wherein the plant is a monocot plant.

[0226] Embodiment 92. The composition of embodiment 91, wherein the plant is a plant from the family Gramineae.

[0227] Embodiment 93. The composition of embodiment 92, wherein the plant is a rice plant (Oryza sps.).

[0228] Embodiment 94. The composition of embodiment 93, wherein the recombinant human transferrin is produced in rice.

[0229] Embodiment 95. The composition of any of embodiments 85-94, wherein the bacteria is chosen from human pathogenic Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae (type B), H. influenzae (non-typeable), Moraxella catarrhalis, extraintestinal pathogenic Esherichia coli, and Salmonella typhimurium SL1344.

[0230] Embodiment 96. The composition of embodiment 95, wherein the bacteria is chosen from human pathogenic Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae (type B), and H. influenzae (non-typeable).

[0231] Embodiment 97. The composition of embodiment 96, wherein the bacteria is human pathogenic Neisseria meningitidis.

[0232] Embodiment 98. The composition of embodiment 97, wherein the human pathogenic Neisseria meningitidis does not express one or more, or two or more, or three or more, or four or more, of porin protein PorA, hexa-acyl lipooligosaccharide (LOS), capsular polysaccharide, and sialylated LOS.

[0233] Embodiment 99. The composition of embodiment 98, wherein the human pathogenic Neisseria meningitidis does not express the porin protein PorA.

[0234] Embodiment 100. The composition of embodiment 98, wherein the human pathogenic Neisseria meningitidis does not express hexa-acyl lipooligosaccharide (LOS).

[0235] Embodiment 101. The composition of embodiment 98, wherein the human pathogenic Neisseria meningitidis does not express capsular polysaccharide.

[0236] Embodiment 102. The composition of embodiment 98, wherein the human pathogenic Neisseria meningitidis produces penta-acyl lipooligosaccharide (LOS).

[0237] Embodiment 103. The composition of embodiment 97, wherein the human pathogenic Neisseria meningitidis has a knocked out gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and Fllbp.

[0238] Embodiment 104. The composition of embodiment 130, wherein the human pathogenic Neisseria meningitidis has a knocked out or disrupted IpxLl gene.Attorney Docket No. GMV0005-401-PC

[0239] Embodiment 105. The composition of embodiment 103, wherein the human pathogenic Neisseria meningitidis has knocked out or disrupted siaD and GalE genes.

[0240] Embodiment 106. The composition of embodiment 103, wherein the human pathogenic Neisseria meningitidis has a knocked out or disrupted FHbp gene.

[0241] Embodiment 107. The composition of embodiment 97, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of a gene product from a gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.

[0242] Embodiment 108. The composition of embodiment 97, wherein the human pathogenic Neisseria meningitidis has a replacement of wild type (WT) Factor H binding protein (FHbp) subfamily B, siaD-galE, and IpxLl genes with subfamily A FHbp ID22 mutant comprises one or more of L130R, G133D, and K218N substitutions.

[0243] Embodiment 109. The composition of embodiment 97, wherein the subfamily A FHbp ID22 mutant comprises two or more of L130R, G133D, and K218N substitutions.

[0244] Embodiment 110. The composition of embodiment 97, wherein the subfamily A FHbp 1D22 mutant comprises three of L130R, G133D, and K218N substitutions.

[0245] Embodiment 111. The composition of embodiment 97, wherein the human pathogenic Neisseria meningitidis has a replacement of WT FHbp subfamily B, siaD-galE, and IpxLl genes with subfamily B FHbp ID9 single mutant with S228R substitution.

[0246] Embodiment 112. The composition of embodiment 107, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of IpxLl.

[0247] Embodiment 113. The composition of embodiment 107, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of siaD and GalE.

[0248] Embodiment 114. The composition of embodiment 107, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of FHbp.

[0249] Embodiment 115. The composition of embodiment 97, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of a gene product from a gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.

[0250] Embodiment 116. The composition of embodiment 115, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of IpxLl.

[0251] Embodiment 117. The composition of embodiment 115, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of siaD and GalE.Attorney Docket No. OMV0005-401-PC

[0252] Embodiment 118. The composition of embodiment 115, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of FHbp.

[0253] Embodiment 119. The composition of embodiment 97, wherein the human pathogenic Neisseria meningitidis does not express sialylated LOS.

[0254] Embodiment 120. The composition of embodiment 97, wherein the human pathogenic Neisseria meningitidis has a reduced ability to bind human Factor H.

[0255] Embodiment 121. The composition of embodiment 120, wherein the human pathogenic Neisseria meningitidis has a reduced ability to bind human Factor H due to a replacement of the factor H binding protein gene with DNA coding for a Factor H binding protein gene expressing a mutant Factor H binding protein with a reduced ability to bind human Factor H.

[0256] Embodiment 122. The composition of any of embodiments 97 to 121, wherein the human pathogenic Neisseria meningitidis is transformed with a multicopy plasmid that contains an engineered promoter for overexpressing a mutant Factor H binding protein with reduced Factor H binding.

[0257] Embodiment 123. The composition of embodiment 97, wherein the human pathogenic Neisseria meningitidis has at least two of, or at least three of, or at least four of, or at least five of, or all of:a) does not express porin protein PorA;b) does not express capsular polysaccharide;c) does not express sialylated LOS;d) produces penta- acyl lipooligosaccharide instead of hexa-acyl LOS; e) has a replacement of the factor II binding protein gene with DNA coding for a Factor H binding protein gene expressing a mutant Factor H binding protein with reduced ability to bind human Factor H; andf) is transformed with a multicopy plasmid that contains an engineered promoter for overexpressing a mutant Factor H binding protein with reduced Factor H binding.

[0258] Embodiment 124. The composition of any of embodiments 97 to 123, wherein the human pathogenic Neisseria meningitidis is H44 / 76.

[0259] Embodiment 125. The composition of any of embodiments 97 to 124, wherein the bacteria are grown in chemically defined culture medium comprising recombinant human transferrin.Attorney Docket No. GMV0005-401-PC

[0260] Embodiment 126. The composition of embodiment 125, wherein the chemically defined culture medium is chosen from Catlin 6 medium, gonococcal (GC) medium, Frantz medium, modified Frantz medium, Muller-Hinton medium, Thayer-Martin agar, modified Thayer-Martin agar, New York City (NYC) agar, chocolate agar, and GC agar base.

[0261] Embodiment 127. The composition of embodiment 126, wherein the chemically defined culture medium is Catlin 6 medium.

[0262] Embodiment 128. The composition of embodiment 126, wherein the chemically defined culture medium is supplemented with glucose, D, L-lactate, cysteine, cystine, or combinations thereof.

[0263] Embodiment 129. The composition of any of embodiments 125 to 128, wherein the chemically defined culture medium is supplemented with recombinant human transferrin.

[0264] Embodiment 130. The composition of any of embodiments 125 to 129, wherein the chemically defined culture medium comprises between about 0.5 and about 5 g / L recombinant human transferrin.

[0265] Embodiment 131. The composition of embodiment 130, wherein the chemically defined culture medium comprises between about 0.75 and about 2 g / L recombinant human transferrin.

[0266] Embodiment 132. The composition of embodiment 131, wherein the chemically defined culture medium comprises between about 1.25 and about 2 g / L recombinant human transferrin.

[0267] Embodiment 133. The composition of any of embodiments 125 to 132, wherein the chemically defined culture medium comprises about 0.75, about 1, about 1.25, about 1.5, about 1.75, or about 2 g / L recombinant human transferrin.

[0268] Embodiment 134. The composition of embodiment 133, wherein the chemically defined culture medium comprises about 1.5 g / L recombinant human transferrin.

[0269] Embodiment 135. The composition of embodiment 96, wherein the bacteria is human pathogenic Neisseria gonorrhoeae.

[0270] Embodiment 136. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae has decreased or knocked-out expression and / or function of the lipid A biosynthesis lauroyl acyltransferase (IpxLl) gene and reduction of expression of the modifiable protein (rmp) gene.Attorney Docket No. GMV0005-401-PC

[0271] Embodiment 137. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out gene selected from one or more, or two or more, of IpxLl and rmp.

[0272] Embodiment 138. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out or disrupted IpxLl gene.

[0273] Embodiment 139. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out or disrupted rmp gene.

[0274] Embodiment 140. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of a gene product from a gene selected from one or more, or two or more, of IpxLl and rmp.

[0275] Embodiment 141. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of IpxLl.

[0276] Embodiment 142. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of rmp.

[0277] Embodiment 143. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of a gene product from a gene selected from one or more, or two or more, of IpxLl and rmp.

[0278] Embodiment 144. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of IpxLl.

[0279] Embodiment 145. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of rmp.

[0280] Embodiment 146. The composition of embodiment 135, wherein the human pathogenic Neisseria gonorrhoeae strain is chosen from FA1090, WIIO F, WIIO G, WIIO N, WHO M, and MSI 1.

[0281] Embodiment 147. The composition of embodiment 135, wherein the chemically defined culture medium is GCLB medium.

[0282] Embodiment 148. The composition of embodiment 147, wherein the chemically defined culture medium is supplemented with Peptone #3 from an animal source or Veggie™ Peptone derived from a vegetable source, or combinations thereof.

[0283] Embodiment 149. The composition of embodiment 135, wherein the chemically defined culture medium is supplemented with recombinant human transferrin.Attorney Docket No. OMV0005-401-PC

[0284] Embodiment 150. The composition of embodiment 149, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin results in at least 3-fold higher densities of bacteria.

[0285] Embodiment 151. The composition of embodiment 150, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin results in at least 5-fold higher production of OMVs.

[0286] Embodiment 152. The composition of embodiment 149, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 6 to about 16 hours.

[0287] Embodiment 153. The composition of embodiment 152, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 10 to about 14 hours.

[0288] Embodiment 154. The composition of embodiment 153, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 12 hours.

[0289] Embodiment 155. The composition of embodiment 149, wherein the culture medium comprises between about 0.5 and about 5 g / L recombinant human transferrin.

[0290] Embodiment 156. The composition of embodiment 155, wherein the culture medium comprises between about 0.75 and about 2 g / L recombinant human transferrin.

[0291] Embodiment 157. The composition of embodiment 156, wherein the culture medium comprises between about 1.25 and about 2 g / L recombinant human transferrin.

[0292] Embodiment 158. The composition of embodiment 157, wherein the culture medium comprises about 0.75, about 1, about 1.25, about 1.5, about 1.75, or about 2 g / L recombinant human transferrin.

[0293] Embodiment 159. The composition of embodiment 158, wherein the culture medium comprises about 1.5 g / L recombinant human transferrin.

[0294] Embodiment 160. The composition of any of embodiments 85-159, wherein the OMVs are native OMVs (NOMVs) blebbed from mutant strains of Neisseria.

[0295] Embodiment 161. The composition of any of embodiments 85-159, wherein the OMVs are produced by genetic, chemical or mechanical methods from mutant strains of Neisseria.

[0296] Embodiment 162. A pharmaceutical composition comprising purified outer membrane vesicles (OMVs) made by the process of any of embodiments 1-84 and a therapeutically acceptable excipient.Attorney Docket No. GMV0005-401-PC

[0297] Embodiment 163. The pharmaceutical composition of embodiment 162, wherein the OMVs comprise at least one type of gonococcal recombinant lipoprotein.

[0298] Embodiment 164. An immunogenic composition produced by the process of any of embodiments 1- 84, comprising outer membrane vesicles (OMVs) obtained from mutant strains of Neisseria, comprising:a) OMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of wild type (WT) Factor H binding protein (FHbp) subfamily B, siaD-galE, and IpxLl genes with subfamily A FHbp 1D22 mutant comprises one or more of L130R, G133D, and K218N substitutions; andb) OMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of WT FHbp subfamily B, siaD-galE, and IpxLl genes with subfamily B FHbp ID9 single mutant with S228R substitution..

[0299] Embodiment 165. An immunogenic composition comprising outer membrane vesicles (OMVs) obtained from mutant strains of Neisseria, comprising:a) OMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of wild type (W T) Factor H binding protein (FHbp) subfamily B, siaD-galE, and IpxLl genes with subfamily A FHbp ID22 mutant comprises one or more of L130R, G133D, and K218N substitutions; andb) OMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of WT FHbp subfamily B, siaD-galE, and IpxLl genes with subfamily B FHbp ID9 single mutant with S228R substitution; andc) OMVs from a Neisseria gonorrhoeae parent strain with decreased or knocked-out expression and / or function of the lipid A biosynthesis lauroyl acyltransferase (IpxLl) gene and reduction of expression of the modifiable protein (rmp) gene.

[0300] Embodiment 166. The immunogenic composition of either of embodiments 164-165, wherein the subfamily A FHbp ID22 mutant comprises two or more of L130R, G133D, and K218N substitutions.

[0301] Embodiment 167. The immunogenic composition of any one of embodiments 164-166, wherein the subfamily A FHbp ID22 mutant comprises three of L130R, G133D, and K218N substitutions.

[0302] Embodiment 168. The immunogenic composition of any one of embodiments 164-167, wherein the substitutions to wild type FHbp produce a modified FHbp that has reduced binding to human Factor H.Attorney Docket No. GMV0005-401-PC

[0303] Embodiment 169. The immunogenic composition of any one of embodiments 164-168, wherein the NOMV contains four copies of the modified genes that results in overexpression of the FHbp.

[0304] Embodiment 170. The immunogenic composition of any one of embodiments 164-169, wherein the disruption of IpxLl gene results in penta- acylated lipo-oligosaccharides.

[0305] Embodiment 171. The immunogenic composition of any one of embodiments 164-170, wherein the disruption of the siaD-GalE genes eliminates sialic acid-containing capsular polysaccharides and LOS antigens.

[0306] Embodiment 172. The immunogenic composition of any one of embodiments 164-171, wherein both H44 / 76 strains comprise a copy of the recombinant FHbp gene inserted into the fhbp gene, the siaD-galE locus, and the IpxLl gene.

[0307] Embodiment 173. The immunogenic composition of any one of embodiments 164-172, wherein the H44 / 76 strains further comprise a plasmid with a 4th copy of the recombinant FHbp gene.

[0308] Embodiment 174. The immunogenic composition of any one of embodiments 164-173, wherein the ID22 triple mutant strain and ID9 single mutant strain are present in a 70:30 ratio of ID22: ID9.

[0309] Embodiment 175. The immunogenic composition of any one of embodiments 164-174, wherein the composition is adsorbed to aluminum hydroxide adjuvant.

[0310] Embodiment 176. The immunogenic composition of any one of embodiments 164-165, wherein the parent strain of b) is chosen from FA1090, WHO F, WHO G, WHO N, WHO M, and MS11.

[0311] Embodiment 177. The immunogenic composition of any one of embodiments 164-165 and 176, wherein the parent strain of b) has the reduction modifiable protein (rmp) gene completely knocked out.

[0312] Embodiment 178. The immunogenic composition of any one of embodiments 164-165 and 176-177, wherein the parent strain of b) has the lipid A biosynthesis lauroyl acyltransferase (Ipxl 1) gene knocked out.

[0313] Embodiment 179. The immunogenic composition of embodiment 14, wherein the first 178 amino acids of the Ipxl 1 gene are present.

[0314] Embodiment 180. The immunogenic composition of any one of embodiments 164-165 and 176-179, wherein the parent strain of b) further comprises a knockout of the one or more of the OpaD, Opa54, Opa60, and Opas genes.Attorney Docket No. OMV0005-401-PC

[0315] Embodiment 181. The immunogenic composition of any one of embodiments 164-180, wherein the composition is administered via intramuscular injection or intranasal administration.

[0316] Embodiment 182. The immunogenic composition of any one of embodiments 164-181, wherein the composition is not pyrogenic.

[0317] Embodiment 183. A method of eliciting a bi-valent antibody response, comprising administration of the immunogenic composition of any one of embodiments 165-182.

[0318] Embodiment 184. The method of embodiment 183, wherein the response elicits antibodies specific for the vaccine antigen present in each component as an NOMV-FHbp or NOMV-Ng bi-valent vaccine or when combined as a tetra- valent combination vaccine (NOMV-combo).

[0319] Embodiment 185. A method of eliciting serum bactericidal activity (SBA) against diverse McnB and Ng strains and against both diverse meningococcal and gonococcal strains, comprising administration of the immunogenic composition of any one of embodiments 165-182.

[0320] Embodiment 186. A method of inhibiting Neisseria gonorrhoeae and Neisseria meningitidis colonization of cervical and vaginal cells, comprising administration of the immunogenic composition of any one of embodiments 165-182.

[0321] Embodiment 187. A method of vaccinating a patient against Neisseria gonorrhoeae and Neisseria meningitidis infections comprising injecting an effective amount of the immunogenic composition of any one of embodiments 165-182.

[0322] Embodiment 188. The method of any of embodiments 183-187, wherein the OMVs are native OMVs (NOMVs) blebbed from mutant strains of Neisseria.

[0323] Embodiment 189. The method of any of embodiments 183-187, wherein the OMVs are produced by genetic, chemical or mechanical methods from mutant strains of Neisseria.

[0324] The detailed description set-forth above is provided to aid those skilled in the art in practicing the present disclosure. However, the disclosure described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of one or more aspects of the disclosure. Any equivalent embodiments are intended to be within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein willAttorney Docket No. OMV0005-401-PCbecome apparent to those skilled in the art from the foregoing description, which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also, unless otherwise indicated, intended to fall within the scope of the appended claims

Claims

Attorney Docket No. OMV0005-401-PCCLAIMSWhat is claimed is:

1. A process for producing outer membrane vesicles (OMVs) from bacteria that acquire iron from human transferrin comprising growing the bacteria in medium comprising recombinant human transferrin.

2. The process of claim 1, wherein the process comprises:a) growing the bacteria in an atmosphere of ambient air supplemented with 3 % - 5 % CO2;b) isolating individual cultures of bacteria and dispersing the cultures in medium comprising recombinant human transferrin;c) growing the redispersed cultures at about 37 °C;d) adding fresh medium and further growing the cultures at about 37 °C in an atmosphere of ambient air supplemented with CO2one or more times; and e) purifying the cultures.

3. The process of either of claims 1 and 2, wherein the medium contains negligible detergent or is detergent free.

4. The process of any of claims 1-3, wherein the OMVs are free of EDTA.

5. The process of any of claims 1-4, wherein the cultures are purified by size exclusion chromatography.

6. The process of any of claims 1 -5, wherein recombinant transferrin is human.

7. The process of any of claims 1-6, wherein the recombinant human transferrin has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:49.

8. The process of any of claims 1-7, wherein the recombinant human transferrin comprises a C-lobe which has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:51.

9. The process of any of claims 1-8, wherein the recombinant transferrin is produced in a plant.

10. The process of claim 9, wherein the plant is a monocot plant.

11. The process of claim 10, wherein the plant is a plant from the family Gramineae.

12. The process of claim 11, wherein the plant is a rice plant (Oryza sps.).

13. The process of any of claims 1-12, wherein the recombinant transferrin is produced in rice.Attorney Docket No. OMV0005-401-PC14. The process of any of claims 1-13, wherein the bacteria is chosen from human pathogenic Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae (type B), H. influenzae (non-typeable), Moraxella catarrhalis, extraintestinal pathogenic Esherichia coli, and Salmonella typhimurium SL1344.

15. The process of claim 14, wherein the bacteria is chosen from human pathogenic Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae (type B), and H. influenzae (non-typeable).

16. The process of claim 1, wherein the bacteria is human pathogenic Neisseria meningitidis.

17. The process of claim 16, wherein the human pathogenic Neisseria meningitidis does not express one or more, or two or more, or three or more, or four or more, of porin protein PorA, hexa-acyl lipooligosaccharide (LOS), capsular polysaccharide, and sialylated LOS.

18. The process of claim 17, wherein the human pathogenic Neisseria meningitidis does not express the porin protein PorA.

19. The process of claim 17, wherein the human pathogenic Neisseria meningitidis does not express hexa-acyl lipooligosaccharide (LOS).

20. The process of claim 17, wherein the human pathogenic Neisseria, meningitidis does not express capsular polysaccharide.

21. The process of claim 17, wherein the human pathogenic Neisseria meningitidis produces penta-acyl lipooligosaccharide (LOS).

22. The process of claim 17, wherein the human pathogenic Neisseria meningitidis has a knocked out gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FIIbp.

23. The process of claim 17, wherein the human pathogenic Neisseria meningitidis has a replacement of wild type (WT) Factor H binding protein (FHbp) subfamily B, siaD-galE, and IpxLl genes with subfamily A FHbp ID22 mutant comprises one or more of L130R, G133D, and K218N substitutions.

24. The process of claim 23, wherein the subfamily A FHbp ID22 mutant comprises two or more of L130R, G133D, and K218N substitutions.

25. The process of any one of claims 23-24, wherein the subfamily A FHbp ID22 mutant comprises three of L130R, G133D, and K218N substitutions.

26. The process of claim 17, wherein the human pathogenic Neisseria meningitidis has a replacement of WT FHbp subfamily B, siaD-galE, and IpxLl genes with subfamily B FHbp ID9 single mutant with S228R substitution.Attorney Docket No. GMV0005-401-PC27. The process of claim 22, wherein the human pathogenic Neisseria meningitidis has a knocked out or disrupted IpxLl gene.

28. The process of claim 22, wherein the human pathogenic Neisseria meningitidis has knocked out or disrupted siaD and GalE genes.

29. The process of claim 22, wherein the human pathogenic Neisseria meningitidis has a knocked out or disrupted FHbp gene.

30. The process of claim 16, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of a gene product from a gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.

31. The process of claim 30, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of IpxLl.

32. The process of claim 30, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of siaD and GalE.

33. The process of claim 30, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of FHbp.

34. The process of claim 30, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of a gene product from a gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.

35. The process of claim 16, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of IpxLl.

36. The process of claim 35, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of siaD and GalE.

37. The process of claim 35, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of FHbp.

38. The process of claim 16, wherein the human pathogenic Neisseria meningitidis does not express sialylated LOS.

39. The process of claim 16, wherein the human pathogenic Neisseria meningitidis has a reduced ability to bind human Factor H.

40. The process of claim 39, wherein the human pathogenic Neisseria meningitidis has a reduced ability to bind human Factor H due to a replacement of the factor H binding protein gene with DNA coding for a Factor H binding protein gene expressing a mutant Factor H binding protein with a reduced ability to bind human Factor II.

41. The process of either of any of claims 16 to 40, wherein the human pathogenic Neisseria meningitidis is transformed with a multicopy plasmid that contains an engineeredAttorney Docket No. GMV0005-401-PCpromoter for overexpressing a mutant Factor H binding protein with reduced Factor H binding.

42. The process of claim 16, wherein the human pathogenic Neisseria meningitidis has at least two of, or at least three of, or at least four of, or at least five of, or all of:a) does not express porin protein PorA;b) does not express capsular polysaccharide;c) does not express sialylated LOS;d) produces penta-acyl lipooligosaccharide instead of hexa-acyl LOS; e) has a replacement of the factor H binding protein gene with DNA coding for a Factor H binding protein gene expressing a mutant Factor H binding protein with reduced ability to bind human Factor H; andf) is transformed with a multicopy plasmid that contains an engineered promoter for overexpressing a mutant Factor H binding protein with reduced Factor H binding.

43. The process of any of claims 16 to 42, wherein the human pathogenic Neisseria meningitidis is H44 / 76.

44. The process of any of claims 1 to 43, wherein the human pathogenic Neisseria, meningitidis bacteria cultured in medium comprising human transferrin results in at least 3-fold higher densities of bacteria.

45. The process of claim 44, wherein the human pathogenic Neisseria meningitidis bacteria cultured in medium comprising human transferrin results in at least 5-fold higher production of OMVs.

46. The process of any of claims 1 to 45, wherein the human pathogenic Neisseria meningitidis bacteria cultured in medium comprising human transferrin is grown for about 6 to about 16 hours.

47. The process of claim 46, wherein the human pathogenic Neisseria meningitidis bacteria cultured in medium comprising human transferrin is grown for about 10 to about 14 hours.

48. The process of claim 47, wherein the human pathogenic Neisseria meningitidis bacteria cultured in chemically defined medium comprising human transferrin is grown for about 12 hours.

49. The process of any of claims 1 to 48, wherein the medium is chosen from Catlin 6 medium, gonococcal (GC) medium, Frantz medium, modified Frantz medium, Muller-HintonAttorney Docket No. GMV0005-401-PCmedium, Thayer-Martin agar, modified Thayer-Martin agar, New York City (NYC) agar, chocolate agar, and GC agar base.

50. The process of claim 49, wherein the medium is Catlin 6 medium.

51. The process of claim 49, wherein the chemically defined culture medium is supplemented with glucose, D, L-lactate, cysteine, cystine, or combinations thereof.

52. The process of any of claims 1 to 51, wherein the chemically defined culture medium is supplemented with recombinant human transferrin.

53. The process of any of claims 1 to 52, wherein the chemically defined culture medium comprises between about 0.5 and about 5 g / L recombinant human transferrin.

54. The process of claim 53, wherein the chemically defined culture medium comprises between about 0.75 and about 2 g / L recombinant human transferrin.

55. The process of claim 54, wherein the chemically defined culture medium comprises between about 1.25 and about 2 g / L recombinant human transferrin.

56. The process of any of claims 1 to 55, wherein the chemically defined culture medium comprises about 0.75, about 1, about 1.25, about 1.5, about 1.75, or about 2 g / L recombinant human transferrin.

57. The process of claim 56, wherein the chemically defined culture medium comprises about 1.5 g / L recombinant human transferrin.

58. The process of claim 15, wherein the bacteria is human pathogenic Neisseria gonorrhoeae.

59. The process of claim 58, wherein the human pathogenic Neisseria gonorrhoeae has decreased or knocked-out expression and / or function of the lipid A biosynthesis lauroyl acyltransferase (IpxLl) gene and reduction of expression of the modifiable protein (rmp) gene.

60. The process of claim 59, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out gene selected from one or more, or two or more, of IpxLl and rmp.

61. The process of claim 60, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out or disrupted IpxLl gene.

62. The process of claim 61, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out or disrupted rmp gene.

63. The process of claim 58, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of a gene product from a gene selected from one or more, or two or more, of IpxLl and rmp.Attorney Docket No. GMV0005-401-PC64. The process of claim 63, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of IpxLl.

65. The process of claim 63, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of rmp.

66. The process of claim 58, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of a gene product from a gene selected from one or more, or two or more, of IpxLl and rmp.

67. The process of claim 66, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of IpxLl.

68. The process of claim 66, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of rmp.

69. The process of any of claims 58-68, wherein the human pathogenic Neisseria gonorrhoeae strain is chosen from FA1090, WHO F, WHO G, WHO N, WHO M, and MS11.

70. The process of any of claims 58-69, wherein the chemically defined culture medium is GCLB medium.

71. The process of claim 70, wherein the chemically defined culture medium is supplemented with Peptone #3 from an animal source or Veggie™ Peptone derived from a vegetable source, or combinations thereof.

72. The process of any of claims 58-71, wherein the chemically defined culture medium is supplemented with recombinant human transferrin.

73. The process of any of claims 58-72, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin results in at least 3-fold higher densities of bacteria.

74. The process of claim 73, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin results in at least 5-fold higher production of OMVs.

75. The process of any of claims 58-74, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 6 to about 16 hours.

76. The process of claim 75, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 10 to about 14 hours.Attorney Docket No. GMV0005-401-PC77. The process of claim 76, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 12 hours.

78. The process of any of claims 58-77, wherein the culture medium comprises between about 0.5 and about 5 g / L recombinant human transferrin.

79. The process of claim 78, wherein the culture medium comprises between about 0.75 and about 2 g / L recombinant human transferrin.

80. The process of claim 79, wherein the culture medium comprises between about 1.25 and about 2 g / L recombinant human transferrin.

81. The process of any of claims 58-80, wherein the culture medium comprises about 0.75, about 1, about 1.25, about 1.5, about 1.75, or about 2 g / L recombinant human transferrin.

82. The process of claim 81, wherein the culture medium comprises about 1.5 g / L recombinant human transferrin.

83. The process of any of claims 1-82, wherein the OMVs are native OMVs (NOMVs) blebbed from mutant strains of Neisseria.

84. The process of any of claims 1-82, wherein the OMVs are produced by genetic, chemical or mechanical methods from mutant strains of Neisseria.

85. A composition comprising outer membrane vesicles (OMVs), bacteria that acquire iron from human transferrin, and recombinant human transferrin.

86. The composition of claim 85, wherein the medium contains negligible detergent or is detergent free.

87. The composition of claim 85, wherein the OMVs are free of EDTA.

88. The composition of any of claims 85-87, wherein the recombinant human transferrin has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:

49.

89. The composition of any of claims 85-88, wherein the recombinant human transferrin comprises a C-lobe which has a sequence at least 80%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:51.

90. The composition of any of claims 85-89, wherein the recombinant human transferrin is produced in a plant.

91. The composition of claim 90, wherein the plant is a monocot plant.

92. The composition of claim 91, wherein the plant is a plant from the family Gramineae.

93. The composition of claim 92, wherein the plant is a rice plant (Oryza sps.).

94. The composition of claim 93, wherein the recombinant human transferrin is produced in rice.Attorney Docket No. GMV0005-401-PC95. The composition of any of claims 85-94, wherein the bacteria is chosen from human pathogenic Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae (type B), H. influenzae (non-typeable), Moraxella catarrhalis, extraintestinal pathogenic Esherichia coli, and Salmonella typhimurium SL1344.

96. The composition of claim 95, wherein the bacteria is chosen from human pathogenic Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae (type B), and H. influenzae (non-typeable).

97. The composition of claim 96, wherein the bacteria is human pathogenic Neisseria meningitidis.

98. The composition of claim 97, wherein the human pathogenic Neisseria meningitidis does not express one or more, or two or more, or three or more, or four or more, of porin protein PorA, hexa-acyl lipooligosaccharide (LOS), capsular polysaccharide, and sialylated LOS.

99. The composition of claim 98, wherein the human pathogenic Neisseria meningitidis does not express the porin protein PorA.

100. The composition of claim 98, wherein the human pathogenic Neisseria meningitidis does not express hexa-acyl lipooligosaccharide (LOS).

101. The composition of claim 98, wherein the human pathogenic Neisseria meningitidis does not express capsular polysaccharide.

102. The composition of claim 98, wherein the human pathogenic Neisseria meningitidis produces penta-acyl lipooligosaccharide (LOS).

103. The composition of claim 97, wherein the human pathogenic Neisseria meningitidis has a knocked out gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.

104. The composition of claim 130, wherein the human pathogenic Neisseria meningitidis has a knocked out or disrupted IpxLl gene.

105. The composition of claim 103, wherein the human pathogenic Neisseria meningitidis has knocked out or disrupted siaD and GalE genes.

106. The composition of claim 103, wherein the human pathogenic Neisseria meningitidis has a knocked out or disrupted FHbp gene.

107. The composition of claim 97, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of a gene product from a gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.Attorney Docket No. GMV0005-401-PC108. The composition of claim 97, wherein the human pathogenic Neisseria meningitidis has a replacement of wild type (WT) Factor H binding protein (FHbp) subfamily B, siaD-galE, and IpxLl genes with subfamily A FHbp ID22 mutant comprises one or more of L130R, G133D, and K218N substitutions.

109. The composition of claim 97, wherein the subfamily A FHbp ID22 mutant comprises two or more of L130R, G133D, and K218N substitutions.

110. The composition of claim 97, wherein the subfamily A FHbp ID22 mutant comprises three of L130R, G133D, and K218N substitutions.

111. The composition of claim 97, wherein the human pathogenic Neisseria meningitidis has a replacement of WT FHbp subfamily B, siaD-galE, and IpxLl genes with subfamily B FHbp ID9 single mutant with S228R substitution.

112. The composition of claim 107, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of IpxLl.

113. The composition of claim 107, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of siaD and GalE.

114. The composition of claim 107, wherein the human pathogenic Neisseria meningitidis expresses a reduced amount of FHbp.

115. The composition of claim 97, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of a gene product from a gene selected from one or more, or two or more, or three or more, or four or more, of IpxLl, siaD, GalE, and FHbp.

116. The composition of claim 115, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of IpxLl.

117. The composition of claim 115, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of siaD and GalE.

118. The composition of claim 115, wherein the human pathogenic Neisseria meningitidis expresses a negligible amount of FHbp.

119. The composition of claim 97, wherein the human pathogenic Neisseria meningitidis does not express sialylated LOS.

120. The composition of claim 97, wherein the human pathogenic Neisseria meningitidis has a reduced ability to bind human Factor H.

121. The composition of claim 120, wherein the human pathogenic Neisseria meningitidis has a reduced ability to bind human Factor II due to a replacement of the factor II binding protein gene with DNA coding for a Factor H binding protein gene expressing a mutant Factor H binding protein with a reduced ability to bind human Factor H.Attorney Docket No. OMV0005-401-PC122. The composition of any of claims 97 to 121, wherein the human pathogenic Neisseria meningitidis is transformed with a multicopy plasmid that contains an engineered promoter for overexpressing a mutant Factor H binding protein with reduced Factor H binding.

123. The composition of claim 97, wherein the human pathogenic Neisseria meningitidis has at least two of, or at least three of, or at least four of, or at least five of, or all of:a) does not express porin protein PorA;b) does not express capsular polysaccharide;c) does not express sialylated LOS;d) produces penta-acyl lipooligosaccharide instead of hexa-acyl LOS; e) has a replacement of the factor H binding protein gene with DNA coding for a Factor H binding protein gene expressing a mutant Factor H binding protein with reduced ability to bind human Factor H; andf) is transformed with a multicopy plasmid that contains an engineered promoter for overexpressing a mutant Factor H binding protein with reduced Factor H binding.

124. The composition of any of claims 97 to 123, wherein the human pathogenic Neisseria meningitidis is H44 / 76.

125. The composition of any of claims 97 to 124, wherein the bacteria are grown in chemically defined culture medium comprising recombinant human transferrin.

126. The composition of claim 125, wherein the chemically defined culture medium is chosen from Catlin 6 medium, gonococcal (GC) medium, Frantz medium, modified Frantz medium, Muller-Hinton medium, Thayer-Martin agar, modified Thayer-Martin agar, New York City (NYC) agar, chocolate agar, and GC agar base.

127. The composition of claim 126, wherein the chemically defined culture medium is Catlin 6 medium.

128. The composition of claim 126, wherein the chemically defined culture medium is supplemented with glucose, D, L-lactate, cysteine, cystine, or combinations thereof.

129. The composition of any of claims 125 to 128, wherein the chemically defined culture medium is supplemented with recombinant human transferrin.

130. The composition of any of claims 125 to 129, wherein the chemically defined culture medium comprises between about 0.5 and about 5 g / L recombinant human transferrin.

131. The composition of claim 130, wherein the chemically defined culture medium comprises between about 0.75 and about 2 g / L recombinant human transferrin.Attorney Docket No. OMV0005-401-PC132. The composition of claim 131, wherein the chemically defined culture medium comprises between about 1.25 and about 2 g / L recombinant human transferrin.

133. The composition of any of claims 125 to 132, wherein the chemically defined culture medium comprises about 0.75, about 1, about 1.25, about 1.5, about 1.75, or about 2 g / L recombinant human transferrin.

134. The composition of claim 133, wherein the chemically defined culture medium comprises about 1.5 g / L recombinant human transferrin.

135. The composition of claim 96, wherein the bacteria is human pathogenic Neisseria gonorrhoeae.

136. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae has decreased or knocked-out expression and / or function of the lipid A biosynthesis lauroyl acyltransferase (IpxLl) gene and reduction of expression of the modifiable protein (imp) gene.

137. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out gene selected from one or more, or two or more, of IpxLl and rmp.

138. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out or disrupted IpxLl gene.

139. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae has a knocked out or disrupted rmp gene.

140. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of a gene product from a gene selected from one or more, or two or more, of IpxLl and rmp.

141. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of IpxLl.

142. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a reduced amount of rmp.

143. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of a gene product from a gene selected from one or more, or two or more, of IpxLl and rmp.

144. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of IpxLl.

145. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae expresses a negligible amount of rmp.Attorney Docket No. GMV0005-401-PC146. The composition of claim 135, wherein the human pathogenic Neisseria gonorrhoeae strain is chosen from FA 1090, WHO F, WHO G, WHO N, WHO M, and MSI 1.

147. The composition of claim 135, wherein the chemically defined culture medium is GCLB medium.

148. The composition of claim 147, wherein the chemically defined culture medium is supplemented with Peptone #3 from an animal source or Veggie™ Peptone derived from a vegetable source, or combinations thereof.

149. The composition of claim 135, wherein the chemically defined culture medium is supplemented with recombinant human transferrin.

150. The composition of claim 149, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin results in at least 3 -fold higher densities of bacteria.

151. The composition of claim 150, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin results in at least 5 -fold higher production of OMVs.

152. The composition of claim 149, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 6 to about 16 hours.

153. The composition of claim 152, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 10 to about 14 hours.

154. The composition of claim 153, wherein the human pathogenic Neisseria gonorrhoeae bacteria cultured in medium comprising human transferrin is grown for about 12 hours.

155. The composition of claim 149, wherein the culture medium comprises between about 0.5 and about 5 g / L recombinant human transferrin.

156. The composition of claim 155, wherein the culture medium comprises between about 0.75 and about 2 g / L recombinant human transferrin.

157. The composition of claim 156, wherein the culture medium comprises between about 1.25 and about 2 g / L recombinant human transferrin.

158. The composition of claim 157, wherein the culture medium comprises about 0.75, about 1, about 1.25, about 1.5, about 1.75, or about 2 g / L recombinant human transferrin.

159. The composition of claim 158, wherein the culture medium comprises about 1.5 g / L recombinant human transferrin.Attorney Docket No. GMV0005-401-PC160. The composition of any of claims 85-159, wherein the OMVs are native OMVs (NOMVs) blebbed from mutant strains of Neisseria.

161. The composition of any of claims 85-159, wherein the OMVs are produced by genetic, chemical or mechanical methods from mutant strains of Neisseria.

162. A pharmaceutical composition comprising purified outer membrane vesicles (OMVs) made by the process of any of claims 1-84 and a therapeutically acceptable excipient.

163. The pharmaceutical composition of claim 162, wherein the OMVs comprise at least one type of gonococcal recombinant lipoprotein.

164. An immunogenic composition produced by the process of any of claims 1- 84, comprising outer membrane vesicles (OMVs) obtained from mutant strains of Neisseria, comprising:c) OMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of wild type (WT) Factor H binding protein (FHbp) subfamily B, siaD-galE, and IpxLl genes with subfamily A FHbp ID22 mutant comprises one or more of L130R, G133D, and K218N substitutions; andd) OMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of WT FHbp subfamily B, siaD-galE, and IpxLl genes with subfamily B FHbp ID9 single mutant with S228R substitution..

165. An immunogenic composition comprising outer membrane vesicles (OMVs) obtained from mutant strains of Neisseria, comprising:e) OMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of wild type (WT) Factor H binding protein (FHbp) subfamily B, siaD-galE, and IpxLl genes with subfamily A FHbp ID22 mutant comprises one or more of L130R, G133D, and K218N substitutions; andf) OMVs from a Neisseria meningitidis H44 / 76 parent strain with replacement of WT FHbp subfamily B, siaD-galE, and IpxLl genes with subfamily B FHbp ID9 single mutant with S228R substitution; andg) OMVs from a Neisseria gonorrhoeas parent strain with decreased or knocked-out expression and / or function of the lipid A biosynthesis lauroyl acyltransferase (IpxLl) gene and reduction of expression of the modifiable protein (rmp) gene.

166. The immunogenic composition of either of claims 164-165, wherein the subfamily A FHbp ID22 mutant comprises two or more of L130R, G133D, and K218N substitutions.

167. The immunogenic composition of any one of claims 164-166, wherein the subfamily A FHbp ID22 mutant comprises three of L130R, G133D, and K218N substitutions.Attorney Docket No. GMV0005-401-PC168. The immunogenic composition of any one of claims 164-167, wherein the substitutions to wild type FHbp produce a modified FHbp that has reduced binding to human Factor H.

169. The immunogenic composition of any one of claims 164-168, wherein the NOMV contains four copies of the modified genes that results in overexpression of the FHbp.

170. The immunogenic composition of any one of claims 164-169, wherein the disruption of IpxLl gene results in penta-acylated lipo-oligosaccharides.

171. The immunogenic composition of any one of claims 164-170, wherein the disruption of the siaD-GalE genes eliminates sialic acid-containing capsular polysaccharides and LOS antigens.

172. The immunogenic composition of any one of claims 164-171, wherein both H44 / 76 strains comprise a copy of the recombinant FHbp gene inserted into the fhbp gene, the siaD-galE locus, and the IpxLl gene.

173. The immunogenic composition of any one of claims 164-172, wherein the H44 / 76 strains further comprise a plasmid with a 4th copy of the recombinant FHbp gene.

174. The immunogenic composition of any one of claims 164-173, wherein the 1D22 triple mutant strain and ID9 single mutant strain are present in a 70:30 ratio of ID22: ID9.

175. The immunogenic composition of any one of claims 164-174, wherein the composition is adsorbed to aluminum hydroxide adjuvant.

176. The immunogenic composition of any one of claims 164-165, wherein the parent strain of b) is chosen from FA 1090, WHO F, WHO G, WHO N, WHO M, and MS 11.

177. The immunogenic composition of any one of claims 164-165 and 176, wherein the parent strain of b) has the reduction modifiable protein (rmp) gene completely knocked out.

178. The immunogenic composition of any one of claims 164-165 and 176-177, wherein the parent strain of b) has the lipid A biosynthesis lauroyl acyltransferase (Ipxl 1) gene knocked out.

179. The immunogenic composition of claim 14, wherein the first 178 amino acids of the Ipxl 1 gene are present.

180. The immunogenic composition of any one of claims 164-165 and 176-179, wherein the parent strain of b) further comprises a knockout of the one or more of the OpaD, Opa54, Opa60, and Opas genes.

181. The immunogenic composition of any one of claims 164-180, wherein the composition is administered via intramuscular injection or intranasal administration.Attorney Docket No. OMV0005-401-PC182. The immunogenic composition of any one of claims 164-181, wherein the composition is not pyrogenic.

183. A method of eliciting a bi-valent antibody response, comprising administration of the immunogenic composition of any one of claims 165-182.

184. The method of claim 183, wherein the response elicits antibodies specific for the vaccine antigen present in each component as an NOMV-FHbp or NOMV-Ng bi-valent vaccine or when combined as a tetra-valent combination vaccine (NOMV-combo).

185. A method of eliciting serum bactericidal activity (SB A) against diverse MenB and Ng strains and against both diverse meningococcal and gonococcal strains, comprising administration of the immunogenic composition of any one of claims 165-182.

186. A method of inhibiting Neisseria gonorrhoeae and Neisseria meningitidis colonization of cervical and vaginal cells, comprising administration of the immunogenic composition of any one of claims 165-182.

187. A method of vaccinating a patient against Neisseria gonorrhoeae and Neisseria meningitidis infections comprising injecting an effective amount of the immunogenic composition of any one of claims 165-182.

188. The method of any of claims 183-187, wherein the OMVs are native OMVs (NOMVs) blebbed from mutant strains of Neisseria.

189. The method of any of claims 183-187, wherein the OMVs are produced by genetic, chemical or mechanical methods from mutant strains of Neisseria.

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