US20050244436A1

US20050244436A1 - Combination Neisserial compositions

Info

Publication number: US20050244436A1
Application number: US10/984,376
Authority: US
Inventors: Marzia Giuliani; Mariagrazia Pizza; Rino Rappuoli
Original assignee: Chiron SRL
Current assignee: Novartis AG
Priority date: 1999-05-19
Filing date: 2004-11-09
Publication date: 2005-11-03
Also published as: CN102580072A; CA2838395A1; US9364528B1; EP2270172A1; BR0010721A; EP2270174A1; JP5622344B2; EP2270173B1; US9610342B2; US20170165346A1; AU783894B2; CY1117239T1; MXPA01011867A; WO2000071725A3; US20160151476A1; EP2270172B1; CA2373236A1; DK2270172T3; AU2009200865A1; NZ536676A

Abstract

Compositions comprising a first biological molecule from a Neisseria bacterium and a second biological molecule from a Neisseria bacterium. The term “biological molecule” includes proteins and nucleic acids. Preferred Neisseria species are N.meningitidis and N.gonorrhoeae.

Description

All documents cited herein are incorporated by reference in their entirety.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent application Ser. No. 09/979,263, filed Nov. 19, 2001, which is a §371 filing from PCT IB00/00828, filed May 19, 2000, which claims priority from GB 9911692.3, filed May 19, 1999, GB 9919705.5, filed Aug. 19, 1999 and GB 0005730.7, filed Mar. 9, 2000, from which applications priority is claimed pursuant to the provisions of 35 U.S.C. §§ 119/120, and which applications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to compositions comprising combinations of biological molecules from Neisseria bacteria, particularly N.meningitidis and N.gonorrhoeae.

BACKGROUND ART

Neisseria meningitidis and Neisseria gonorrhoeae are non-motile, Gram negative diplococci that are pathogenic in humans.
Based on the organism's capsular polysaccharide, 12 serogroups of N.meningitidis have been identified. Group A is the pathogen most often implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in most developed countries. Serogroups W135 and Y are responsible for the rest of the cases in the United States and developed countries.
The meningococcal vaccine currently in use is a tetravalent polysaccharide vaccine composed of serogroups A, C, Y and W135. Meningococcus B remains a problem, however. The polysaccharide approach cannot be used because the menB capsular polysaccharide is a polymer of α(2-8)-linked N-acetyl neuraminic acid that is also present in mammalian tissue. One approach to a menB vaccine uses mixtures of outer membrane proteins (OMPs) To overcome the antigenic variability, multivalent vaccines containing up to nine different porins have been constructed [eg. Poolman J T (1992) Development of a meningococcal vaccine. Infect. Agents Dis. 4:13-28]. Additional proteins to be used in outer membrane vaccines have been the opa and opc proteins, but none of these approaches have been able to overcome the antigenic variability [eg. Ala'Aldeen & Borriello (1996) The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains. Vaccine 14(1):49-53].
Given the propensity for meningococcal disease during non-epidemic periods to be caused by multiple strains or strain variants [Russel et al. (1998) Abstracts of 11th International pathogenic Neisseria conference, page 281] together with frequent temporal shifts in the predominant strains in a community, it seems that a universal meningococcal B vaccine will require more than one antigenic species.

DESCRIPTION OF THE INVENTION

Neisserial protein and nucleotide sequences are disclosed in the following documents:

- WO 99/24578
- WO 99/36544
- WO 99/57280
- WO 97/28273
- WO 96/29412
- WO 95/03413
- Tettelin et al. (2000) Science 287:1809-1815

The present invention provides compositions comprising a first biological molecule from a Neisseria bacterium and a second biological molecule from a Neisseria bacterium. The term “biological molecule” includes proteins and nucleic acids.
The compositions may also comprise further biological molecules, preferably also from Neisseria, that is to say the compositions may comprise two or more biological molecules (eg. 3, 4, 5, 6, 7, 8 etc.), at least two of which are from a Neisseria bacterium (eg. 3, 4, 5, 6, 7, 8 etc.). Such compositions include those comprising (i) two or more different Neisserial proteins, (ii) two or more different Neisserial nucleic acids, or (iii) mixtures of one or more Neisserial protein and one or more Neisserial nucleic acid.
In one preferred embodiment, the first and second biological molecules are from different Neisseria species (eg. one is from N.meningitidis and one is from N.gonorrhoeae), but they may be from the same species. The biological molecules in the compositions may be from different serogroups or strains of the same species, such as from N.meningitidis serogroups A, B or C.
The first biological molecule is preferably selected from ORF 287 of the Neisserial genome. Particular amino acid sequences are shown in FIGS. 1A-1D (SEQ ID NOS:1-4, respectively). These sequences, as well as nucleic acid molecules encoding these sequences, are preferred. In particular, SEQ ID NOS: 1 and 4 are sequences for the ORF 287 protein from N.meningitidis serogroup B. SEQ ID NO:1 herein corresponds to SEQ ID NO:1202 of WO 99/57280; SEQ ID NO:4 herein corresponds to NMB2132 of the serogroup B strain MC58 (Tettelin et al., (2000) Science 287:1809-1815); SEQ ID NO:2 herein is the ORF 287 sequence for N.gonorrhoeae and corresponds to SEQ ID NO:1200 of WO 99/57280; and SEQ ID NO:3 herein is the ORF 287 sequence for N.meningitidis serogroup A and corresponds to SEQ ID NO:1204 of WO 99/57280. The biological molecule is preferably a purified or isolated biological molecule.
The second biological molecule is preferably selected from ORF 961 of the Neisserial genome. Particular amino acid sequences are shown in FIGS. 2A and 2B (SEQ ID NOS:5 and 6, respectively). These sequences, as well as nucleic acid molecules encoding these sequences, are preferred. In particular, SEQ ID NOS:5 and 6 are sequences for the ORF 961 protein from N.meningitidis serogroup B. SEQ ID NO:5 herein corresponds to NMB1994 of the serogroup B strain MC58 (Tettelin et al., (2000) Science 287:1809-1815) and SEQ ID NO:6 herein corresponds to SEQ ID NO:2944 of WO 99/57280. The biological molecule is preferably a purified or isolated biological molecule.
One or both of the first and second biological molecules may be a Neisserial biological molecule which is not specifically disclosed herein, and which may not have been identified, discovered, made available to the public or purified before this patent application was filed.
Details as to how the ORF 287 and 961 molecules can be produced and used can be found from the relevant international applications detailed above and these details need not be repeated here.
SEQ ID NOS:1-6 in the compositions of the invention or nucleic acid encoding the same may be supplemented or substituted with molecules comprising sequences homologous (ie. having sequence identity) to SEQ ID NOS: 1-6 or nucleic acid molecules encoding the same. Depending on the particular sequence, the degree of identity is preferably greater than 50% (eg. 65%, 80%, 90%, or more), and includes mutants and allelic variants. Sequence identity between the proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.
SEQ ID NOS:1-6 in the compositions of the invention or nucleic acid molecules encoding the same may be supplemented or substituted with molecules comprising fragments of SEQ ID NOS:1-6 or nucleic acid molecules encoding the same. Such fragments should comprise at least n consecutive monomers from the molecules and, depending on the particular sequence, n is either (i) 7 or more for protein molecules (eg. 8, 10, 12, 14, 16, 18, 20 or more), preferably such that the fragment comprises an epitope from the sequence, or (ii) 10 or more for nucleic acid molecules ( eg 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
Where the composition includes a protein that exists in different nascent and mature forms, the mature form of the protein is preferably used. For example, the mature form of a protein lacking the signal peptide may be used.
In the case of protein molecules, SEQ ID NOS:1-6 in the compositions of the invention may be supplemented or substituted with an antibody that binds to the protein. This antibody may be monoclonal or polyclonal.
In the case of nucleic acid molecules encoding SEQ ID NOS:1-6, the compositions of the invention may be supplemented or substituted with nucleic acid which can hybridise to the Neisserial nucleic acid, preferably under “high stringency” conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDS solution).
It will be appreciated that any nucleic acid in the compositions can take various forms (eg. single stranded, double stranded, vectors, probes etc.). In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.
In certain embodiments, the composition comprises molecules from different Neisseria species, such as one or more N.meningitidis molecule and one or more N.gonorrhoeae molecule. In some embodiments, the composition may comprise molecules from different serogroups and/or strains of the same species, such as strains A and B of N.meningitidis. Further embodiments comprise mixtures of one or more N.meningitidis molecules from different strains and also one or more N.gonorrhoeae molecules.
Many proteins are relatively conserved between different species, serogroups and strains of N.meningitidis and N.gonorrhoeae. PCT/IB00/00642 includes a more detailed experimental analysis of conserved regions in these proteins. To ensure maximum cross-strain recognition and reactivity, regions of proteins that are conserved between different Neisserial species, serogroups and strains can be used in the compositions of the present invention. The invention therefore provides proteins which comprise stretches of amino acid sequence that are shared across the majority of Neisseria, particularly N.meningitidis and N.gonorrhoeae. Preferably, therefore, the composition comprises a protein comprising a fragment of a Neisserial protein (preferably a protein from SEQ ID NOS:1-6), wherein said fragment consists of n consecutive conserved amino acids. Depending on the particular protein, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). The fragment preferably comprises an antigenic or immunogenic region of the Neisserial protein. A “conserved” amino acid is one that is present in a particular Neisserial protein in at least x % of Neisseria (or, preferably, in at least x % of combined N.meningitidis and N.gonorrhoeae strains). The value of x may be 50% or more eg. 66%, 75%, 80%, 90%, 95% or even 100% (ie. the amino acid is found in the protein in question in all Neisseria). In order to determine whether an amino acid is “conserved” in a particular Neisserial protein, it is necessary to compare that amino acid residue in the sequences of the protein in question from a plurality of different Neisseria (a “reference population”). Suitable definitions of “reference populations” can be found in PCT/IB00/00642. Amino acid sequences of different Neissieriae can easily be compared using computers. This will typically involve the alignment of a number of sequences using an algorithm such as CLUSTAL [Thompson et al. (1994) Nucleic Acids Res 22:4673-4680; Trends Biochem Sci (1998) 23:403-405] or, preferably, PILEUP [part of the GCG Wisconsin package, preferably version 9.0]. Conserved amino acids are readily apparent in a multiple sequence alignment—at the amino acid position in question a majority of the aligned sequences will contain a particular amino acid. Conserved amino acids can be made more visually apparent by using a program such as BOXSHADE [available, for instance, at the NIH on-line], PRETTYBOX [GCG Wisconsin, version 10] or JALVIEW [available on-line at EBI].
Specific compositions according to the invention therefore include those comprising:

- two or more biological molecules selected from SEQ ID NOS:1-6;
- one or more biological molecules selected from SEQ ID NOS:1-4 combined with one or more biological molecules selected from SEQ ID NOS:5 and 6;
- one or more biological molecules selected from SEQ ID NOS:1-6 combined with the NspA protein (as disclosed in W096/29412; see also FIG. 3 herein), preferably in mature form;
- one or more biological molecules selected from SEQ ID NOS:1-6 combined with transferrin binding protein A (ThpA) and/or B (TbpB), such as the ThpA and TbpB disclosed in WO00/25811 (or immunogenic fragments thereof).
- one or more fragments of proteins selected from SEQ ID NOS:1-6, with the fragment preferably comprising a stretch of conserved amino acids;
- a combination of different proteins, wherein the combination as a whole includes one or more proteins that is recognised by each strain in a reference population, although each individual protein in the combination may not itself be recognised by each strain in the reference population ie. each member of a reference population recognises at least one protein in the combination.

The invention also provides the compositions of the invention for use as medicaments (eg. as immunogenic compositions or vaccines) or as diagnostic reagents. It also provides the use of the compositions in the manufacture of: (i) a medicament for treating or preventing infection due to Neisserial bacteria; (ii) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria; and/or (iii) a reagent which can raise antibodies against Neisserial bacteria.
The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of a composition according to the invention.
The invention further provides a process for producing a composition according to the invention, comprising the step of bringing one or more of SEQ ID NOS:1-6 into combination with one or more other of SEQ ID NOS:1-6.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D (SEQ ID NOS:1-4) show representative ORF 268 proteins for use in the subject compositions.
FIGS. 2A-2B (SEQ ID NOS:5 and 6) show representative ORF 961 proteins for use in the subject compositions.
FIG. 3A-3C (SEQ ID NOS:7-14) show sequence variability of NspA from various strains of meningococcus B. These sequences may be used as alternatives to the NspA of WO 96/29412.
FIG. 4A-4B show the binding of polyclonal anti-rNspA by indirect fluorescence flow cytometry to encapsulated and non-encapsulated menB strains.
FIGS. 5A-5E show similar data for encapsulated strains 8047, CU385 & M986 (31A) and non-encapsulated strains BZ232, MC58, NG3/88 & NGB165 (31B).
FIG. 6 shows a model of NspA secondary structure.
FIG. 7A-7B show FACS analysis for ORF 919, 287 and 953 antigens and combinations thereof.

EXAMPLES

Example 1

Trivalent Mixture
Proteins ORF1 (e.g. example 77 of WO99/24578; see also WO99/55873), ‘287’ (e.g. FIG. 21 of WO99/57280) and ‘919’ (e.g. WO99/57280 FIG. 23 therein) were combined and adjuvanted with Al(OH)₃. The proteins were from the 2996 strain of MenB.
This mixture was also combined with a MenC polysaccharide conjugate antigen [e.g. Costantino et al. (1992) Vaccine 10:691-698]. OMVs were used as controls.
The mixture was used in a bactericidal assay against the homologous strain and also heterologous MenB strains. Titres were as follows:

2996 BZ133 BZ232 1000 MC58 NGH38

Trivalent 2048 2048 4 <4 64 4

+MenC 2048 >32000 4 128 1024 128

Control 32765 4096 8192 16384 16384 8192

Example 2

Proteins 287, 919 and 953
Proteins 287, 919 and 953 are disclosed in WO99/57280. These proteins from N.meningitidis serogroup B strain 2996 were expressed and tested in a bactericidal assay against strain 2996, alone and in combinations. OMVs from 2996 were used as a positive control.

Antigen

287 919 953 Control

Titre 8192 2048 128 65536

Combination

287 + 919 287 + 953 919 + 953 287 + 919 + 953

Titre 32000 8192 8192 8192
FIG. 7 shows FACS data for the individual antigens and for the four combinations.
It is evident that the antigen mixtures are more effective than the antigens in isolation and, in particular, that combinations of 919+953 give surprisingly good results.

The individual antigens from 2996 and combinations were also tested against different serogroup A, B & C strains (i.e. heterologous challenge). Bactericidal titres were as follows:



Anti-	Serogroup B (MenB) strains	MenA	MenC

gen	2996	BZ133	BZ232	MC58	NGH38	F6124	C11

287	8192	>4096	256	1024	2048	1024	2048
919	2048	—	1024	—	—	—	—
953	128	—	—	—	—	—	—
287 +	32000	>4096	512	512	1024	512	>2048
919
287 +	8192	>4096	1024	512	2048	2048	>2048
953
919 +	8192	—	8192	—	—	—	—
953
Tri-	8192	>2048	256	—	1024	>2048	>2048
va-
lent
Con-	65536	—	8192	2048	—	2048	32768
trol

It is apparent that the antigen mixtures are useful in conferring cross-strain activity.
In a second set of experiments, titres for the individual antigens were as follows:

Anti- Serogroup B (MenB) strains MenA MenC

gen 2996 BZ133 BZ232 MC58 NGH38 F6124 C11

287 16000 2048 16 512 >2048 64 1024

919 16000 — 2048 — — — —

953 2048 — 16 — — — —
The three proteins used in this example were expressed and used in the following forms:

- (1) Protein 287 was expressed in E.coli as a GST fusion;
- (2) Protein 919 was expressed in E.coli without its leader peptide, without its mature N-terminal cysteine, and without any fusion partners (“919-untag”); and
- (3) Protein 953 was expressed using a histidine tag.

Three immunisations were administered with Freund's adjuvants—the first included CFA, and the final two included IFA.

Example 3

Further Polyvalent Combinations

Further combinations of antigens were tested in CD1 mice:



				Bactericidal
Antigens*	Adjuvant	FACS	ELISA	activity

919-his + Orf4-his +	Freund	+++	+	8192
225-his + Orf40-his
Orf4-L + Orf37-GST +	Freund	+++	+	Bacteriostatic
Orf40-his + 502-his +
8-his
919-untag + 791-his +	Freund	+++	+	4096
792-his
919-untag + 287-GST +	Freund	+++	+	8192
953-his
919-untag + 287-GST	Freund	+++	+	32000
287-GST + 953-his	Freund	+++	+	8192
919-untag + 953-his	Freund	+++	+	8192
919-untag + Orf1-his +	Al(OH)₃	+++	+	2048
287-GST
919-untag + Orf1-his +	Al(OH)₃	+++	+	2048
287-GST + MenC
glycoconj.
Orf-46.1-his + 287-GST	Al(OH)₃	n.d.	+	128

*“his” indicates expression and immunisation with a histidine-tagged protein;
“ORF4-L” is the lipidated form of ORF4;
“GST” indicates expression and immunisation with a GST fusion protein;
“919-untag” is as defined in Example 2;
“MenC glycoconj” is the MenC glycoconjugate described in Example 1.

Further combinations of antigens were tested in guinea pigs:

Bactericidal

Antigens Adjuvant FACS ELISA activity

919-his + 287-GST + Freund + + 4096

953-his + Orf46.1-his

919-untage + 287-GST + Freund + + 4096

953-his

287-GST + 953-his Al(OH)₃ n.d. + 256
Evidently the combinations give excellent immunological results.

Example 4

NspA Combinations
NspA protein is disclosed in WO96/29412, and is represented herein as SEQ ID NOS:7-14. The academic literature disclosure of this protein [Martin et al. (1997) J. Exp. Med 185 1173-1183] reported the protein to be highly conserved between Neisseria strains (99% cross-reactivity of anti-NspA antibodies with 250 meningococcal A, B & C strains) and also efficient protection against deadly challenge with live bacteria. There have also been reports that NspA adsorbed on alum elicits serum meningococcal bactericidal antibody responses in rabbits and monkeys [Martin et al. (1998) Abstracts of 11th International pathogenic Neisseria conference, page 198]. On the basis of these data, rNspA (recombinant NspA) is being developed as a vaccine for the prevention of meningococcal disease caused by all serogroups.
Despite sequence conservation, however, it has surprisingly been discovered that rNspA cell surface epitopes are detected on only 65% of the serogroup B strains tested below, and susceptibility to anti-NspA bactericidal activity is also less than that reported by Martin et al. These results contrast with Martin et al., and suggest that a rNspA-based meningococcal B vaccines will need to be supplemented with additional antigens in order to be effective.
The N.meningitidis strains tested in this example were isolated from patients residing in different countries over a period of more than 30 years (see table on page 17). These strains were selected to be representative of widely divergent ‘clonal’ groups, as defined by multilocus isoenzyme typing [Seiler et al. (1996) Mol. Microbiol. 19:841-856] and/or multilocus sequence typing [Maiden et al. (1998) PNAS USA 95:3140-45]. Strain M7, which is derived from strain NMB, contains a transposon insertion that blocks capsular polysaccharide biosynthesis [Stephens et al. (1991) Infect. Immun. 59:4097-4102], but all the other strains are encapsulated.
Based on the nucleotide sequence in Martin et al. (1997), PCR primers were designed and the NspA gene from strain 8047 was amplified. The sequence, including the promoter region, was cloned into pSK+ plasmid (rNspA). A plasmid pTrc.NspA.1 encoding a protein in which a portion of the signal sequence has been replaced with a poly-histidine tag was also used. Both plasmids were expressed in E.coli strain BL21(DE3) and the proteins were purified. In E.coli, rNspA is secreted, rather than being associated with the outer membrane. The protein was partially purified from the culture medium by precipitation with 55% w/v ammonium sulphate, and had an apparent MW of 18.6 kDa, confirmed by Western Blot.
The two forms of NspA (rNspA and denature His-tage NspA) were injected into 6-week old female CD-1 mice to raise antisera. The ability of these to bind to the surface of N.meningitidis strain B was determined using flow cytometric detection of indirect fluorescence assay [Granoff et al. (1998) J. Immunol. 160:5028-36]. The results for strains NMB and M7 (an acapsulated mutant of NMB) are shown in FIG. 4. As expected, anti-group B polysaccharide mAb SEAM-3 [Granoff et al.] only binds to the encapsulated strain, whereas the positive anti-P1.2 (PorA) control mAb binds to both strains. The antisera raised against rNspA is able to bind both strains. Antisera against the His-tag NspA gave negative results, however. These antisera were also negative for strains 8047, CU385 and M986 (FIGS. 5A-5B), but by Western Blot these antisera gave positive results.
These data suggest that antibodies prepared using His-tag NspA recognise epitopes that are present in denatured NspA, but not native NspA as found on the cell-surface in vivo. In contrast, antibodies prepared against rNspA seem to recognise conformational NspA epitopes.
The flow cytometric assay was applied to the strains shown in the table on page 17. FIGS. 5A-5B show that murine antibodies raised against rNspA bind to the surface of strain 8047 (the strain from which the nspA gene was cloned) and strain CU385, but not to M986. FIGS. 5C-5E show similar negative results for strains BZ232, MC58, NG3/88 and NGP165. In all of these negative cases, however, the anticapsular mAb control was positive.
The table on page 17 summarises the flow cytometry results. Although NspA is reported o be accessible at the surface of all intact N.meningitidis strains tested [Martin et al. (1997) J. Exp. Med 185 1173-1183; Plante et al. (1999) Infect. Immun. 67:2855-61], only 11 of the 17 test strains (65%) reacted with the anti-rNspA sera. There was no apparent relationship between cell-surface expression in a given strain and classification (by serotype, subtype, or electrophoretic type), or with year or country of isolation.
In an attempt to explain the differences in reactivity with the anti-rNspA sera, the nspA genes from five of the six negative strains (BX232, NG3/88, NGP165, M136 & M986) and from three of the positive strains (8047, CU385 & NG6/88) were sequenced. The sequence for the sixth negative strain (MC58) was already available from the complete genome sequence.
The nspA sequences for all ten strains were highly conserved, varying at most by 5 nucleotides from the prototype sequence of Martin et al. The most variant protein had only 3 amino acid differences (see FIG. 3). With one exception, all of the amino acid variants involved the same respective residues in discrete segments of the protein. These include the signal peptide, which is not present in the mature protein, and two short segments in the 50 C-terminal residues. These differences do not explain the antisera results, as there are examples of identical variant sequences in strains that were positive and those that were negative (compare M136 & 8047; NGP165 & NG6/88; MC58 & CU385).
As neither lack of the gene nor polymorphism explained the antiserum results, the amount of NspA protein in the outer membranes of five strains (8047, CU385 & NG6/88—all positive for anti-rNspA; M986 & M 136—both negative) were tested. Bacterial cell pellets were extracted with lauryl sarcosinate, and the insoluble outer membrane fractions were analysed. An 18.6 kDa band was seen for all five strains, and this was cross-reactive with anti-His-tag-NspA by Western Blot. Thus strain differences in nspA expression also failed to explain the results.
The ability of anti-rNspA to bind to the bacterial cell surface could be influenced by the amount of polysaccharide capsule present. The quantity of capsular polysaccharide produced by the 17 test strains was therefore assessed by inhibition ELISA.
Extracts of capsular polysaccharide were prepared based on a method described by Corn et al. [J. Infect. Dis. (1993) 167:356-64]. Individual bacterial clones were grown to an OD₆₂₀0.5-0.7 in 7 mL of Mueller-Hinton broth. Bacteria were collected by centrifugation at 5000 g for 15 min, washed in 0.6 mL of 10 mM Hepes, pH 8.0, and then resuspended in 0.6 mL of the same buffer containing 10 mM EDTA and incubated at 37° C. for 1 hr. The cells were pelleted at 10,000 g for 1 minute and the relative amount of meningococcal B polysaccharide antigen released into the supernatant was determined by an inhibition ELISA, performed as described by Azmi et al. [Infect. Immun. (1995) 63:1906-13]. The solid phase antigen in the ELISA was meningococcal B polysaccharide-ADH-biotin absorbed to avidin-coated microtiter plates [Granoff et al.]. The meningococcal B polysaccharide-reactive human paraprotein LIP [Azmi et al.] was used as the primary antibody (0.2 μg/ml). In the absence of inhibitor, this concentration of antibody was sufficient to given an OD of 0.7 to 1.0 after 30 minutes incubation with substrate [Azmi et al.]. The titre of polysaccharide released into the supernatant was measured by determining the dilution of supernatant that resulted in 50% inhibition of antibody binding. Controls in this assay included an EDTA extract prepared from the strain M7, which does not produce any capsular polysaccharide, and purified meningococal B polysaccharide. To ensure that all of the capsular polysaccharide was released by the EDTA treatment, the same inhibition ELISA was performed using the cell pellet resuspended in the same buffer and volume as the capsule extract. The observable inhibitory activity from the cell pellet was between 0 and 10% of the activity observed in the capsule extracts with the latter, higher percentage coming from cell pellets of strains that produce the largest amounts of capsule.
The results for each strain are shown in the table on page 17. On average, the six negative anti-rNspA strains produced three-fold more capsular polysaccharide than the eleven positive strains (respective reciprocal geometric mean dilutions of 676 vs. 224, p<0.05). This may explain the results obtained with the antiserum—conceivably, the presence of larger amounts of capsule could interfere with the ability of the anti-rNspA antibody to bind to NspA epitopes which, in strains with lower amounts of capsule, are accessible.
The complement-dependent bactericidal activity of the anti-rNspA antisera were tested using an assay similar to that described by Mandrell et al. [J. Infect. Dis. (1995) 172:1279-89]. The complement source was human serum from a healthy adult with no detectable anti-capsular antibody to group B polysaccharide and no intrinsic bactericidal activity against the test strain. Serum bactericidal titres were defined as the serum dilution resulting in a 50% decrease in CFU/ml after 60 minutes incubation of bacteria in the reaction mixture, compared to the control CFU/ml at time zero.
Typically, bacteria incubated with a negative control antibody showed a 150-200% increase in CFU/ml during the 60 minutes of incubation. The positive control antibody [anti-capsular IgG2a mAb SEAM 12, Granoff et al. ] showed complement-mediated bactericidal activity against all 17 strains. In contrast, the six strains that were negative for anti-rNspA antisera binding by flow assay were resistant, showing no bactericidal or bacteriostatic effects. Ten of the other eleven positive strains were either killed by complement and the antisera (SWZ107, J351, CU385, NG6/88, BZ198, H44/76, NMB & 8047) or were inhibited (H355 & S3446); strain 1000, however, was not affected.
The ability of the anti-rNspA antisera to confer passive protection against meningococcal B bacteremia was tested in infant rats using a method adapted from Saukkonen [J. Infect. Dis. (1988) 158:209-212]. Briefly, 6-7 day old rats were randomly distributed to nursing mothers. Groups of 5-6 animals were challenged IP with 100 μl of approximately 5000 CFU of N.meningitidis group B bacteria. One strain negative for NspA surface epitopes (M986) and one positive strain (8047) were tested, each of which having been passaged three times in infant rats. Immediately before administration, the bacterial suspension was mixed with different dilutions of test or control antibody (positive control: anticapsular mAb; negative control: anti-E.coli). 18 hours after challenge, blood specimens were obtained from the heart. Aliquots were plated onto chocolate agar, and CFU/ml was determined after overnight incubation at 37° C. in 5% CO₂.

The protective activities of the various co-administered antibodies were as follows:



	Dose per rat		Blood culture

	or		Positive/	CFU/ml	CFU/ml
Antibody treatment	serum dilution	Strain	total	(mean × 10⁻³)	(% of control)

Anticapsular mAb	2 μg	M986		0/6	<1	<1
Anti-rNspA	1:5	M986	6/6	44^a	45
Anti-rNspA	1:25	M986	6/6	93^a	95
Anti-E. coli control	1:5	M986	6/6	98^a	—
Anticapsular mAb	2 μg	8047	0/5	<1	<1
Anti-rNspA	1:5	8047	1/6	0.2^b	2
Anti-rNspA	1:25	8047	1/5	0.4^b	4
Anti-E. coli control	1:5	8047	6/6	10^b	—

^ap > 0.5, compared to geometric mean CFU/ml of control rats
^bp < 0.001, compared to geometric mean CFU/ml of control rats

As can be seen, a dose of 2 μg per rat of the positive anticapsular control was protective against both strains. A 1:5 or 1:25 dilution of anti-rNspA antiserum protected against bacteremia caused by strain 8047. Neither dilution was effective in preventing M986 bacteremia, however.
Despite the positive conclusions of Martin et al., therefore, NspA does not seem to be effective in preventing meningococcal B infection. Approximately one third of strains have decreased cell-surface expression of NspA epitopes when grown in vitro, are resistant to anti-NspA induced complement-mediated bacteriolysis, and are resistant to passive antiserum immunisation. These strains produce large amounts of capsular polysaccharide, and would thus be expected to have the greatest virulence. The ability of a vaccine containing only NspA to confer broad protective immunity against meningococcal B thus has to be doubted.
Compositions comprising NspA [FIG. 3] therefore advantageously comprise further antigens. A preferred aspect of the invention is thus a combination of NspA protein with one or more further Neisserial antigens.

Example 5

NspA Fragments
A model of the secondary structure of NspA is shown in FIG. 6, containing eight transmembrane β-strands and 4 surface-exposed connecting loops. This fits the pattern of alternating hydrophobic and hydrophilic amino acids in NspA, which is characteristic of many β-barrel porins [Weiss et al. (1990) FEBS Letts 267:268-272]
The grey shaded areas in the model indicate segments that are >40% identical and >70% similar to encoded amino acid sequences of opacity proteins (Opa) from N.meningitidis, N. gonorrhoeae, N. flavius, N. sicca, and H. influenzae identified in a BLAST search of the non-redundant GenBank CDS. The alternating sequences are predicted amphiphilic β-strands; vertical segments correspond to transmembrane segments; the top of the figure corresponds to surface exposed segments, labelled as loops 1 to 4.
According to Martin et al., the only significant homology between the deduced amino acid sequence of NspA and those of other proteins are weak homologies with the Neisseria opacity protein (Opa) family in two small segments (˜20 amino acids) near the C-terminal end of the protein. However, separate comparisons of the N- and C-termini of NspA with GenBank reveals a high degree of homology (>40% identity and >70% similarity) between NspA and Opa proteins from N.meningitidis, N. gonorrhoeae, N. flavius, N. sicca, and H. influenzae. The Opa proteins are thought to be integral membrane proteins that have eight transmembrane segments and a β-barrel topology in the membrane similar to that of porin [Merker et al. (1997) Mol. Microbiol. 23:281-293]. The presence of NspA in detergent-insoluble membrane preparations indicate that NspA is located in the outer membrane, which would be consistent with the Opa-like membrane topology shown in the model. In addition, the segments of NspA that are most homologous to those of the Opa proteins are the putative transmembrane segments indicated in the shaded areas of FIG. 6.
The opacity proteins of Neisseria can, under certain circumstances, elicit protective antibody. However, problems with limited antibody accessibility of the opacity proteins in encapsulated bacteria, variability of amino acid sequences in exposed loop segments, and phase variation of protein expression during clinical infection, have limited the ability of Opa to elicit protective antibody consistently [Malorny et al. (1998) J. Infect. Dis. 172:1279-89]. In contrast, there appears to be little or no sequence variation in the surface exposed loops of NspA in FIG. 6. However, it was recently reported that a panel of anti-N.meningitidis NspA monoclonal antibodies that reacted with all meningococcal strains tested reacted with only a limited number of N.gonorrhoeae strains, even though the respective amino acid sequences in the two species are 92% identical. When the respective NspA sequences of the meningococcal and gonococcal strains are compared (FIG. 3), all of the respective amino acid differences result in changes in hydrophobicity or charge, and are located in the putative surface exposed connecting loops (FIG. 6). This finding suggests that the connecting loops in NspA, which are highly conserved in N.meningitidis, may be important epitopes for antibodies that bind to native NspA.
These segments of the molecule, therefore, would appear to be of greatest interest with respect to interacting with protective antibody. However, the putative surface loops of NspA are relatively small (10-14 amino acids) compared to, for example, the highly immunogenic external loops of PorA and Opc (24 to 45 amino acids). The shorter length of the loops may limit the accessibility of NspA surface epitopes for binding interactions with serum antibody, especially in the presence of abundant capsular polysaccharide.
Accordingly, the invention provides the fragments of NspA that are exposed on the cell-surface in FIG. 6, namely SSSLGSAKG (SEQ ID NO:15), NYKAPSTDFKLY (SEQ ID NO:16), NRASVDLGGSDSFSQT (SEQ ID NO:17), and NYIGKVNTVKNVRSG (SEQ ID NO:18), and also provides corresponding fragments from allelic variants of NspA. In addition, the invention provides sub-sequences of these fragments, comprising 7 or more contiguous amino acids from the fragments. The invention further provides proteins comprising these fragments. Nucleic acid encoding these fragments and proteins is also provided.
These NspA fragments, proteins comprising the fragments, and nucleic acid, may be used in the compositions of the invention, in particular as substitutes for full-length NspA. In a further aspect, these fragments, proteins and nucleic acids may be used as products in isolation, that is to say they need not exclusively be used in combination with other biological molecules.

It will be appreciated that the invention has been described by means of example only, and that modifications may be made whilst remaining within the spirit and scope of the invention.

Reactivity of anti-rNspA polyclonal antisera with native NspA exposed on the surface of live, encapsulated,

Neisseria meningitidis B bacteria in relation to susceptibility to bacteriolysis and capsular production.

			Polysaccharide
Meningococcal B Strains	NspA Cell	Anti-rNspA	Capsule

			Serologic		Surface	Bactericidal	Production
Strain	Country	Year	Classification	ET Complex	Reactivity^c	Activity^d(l/titer)	(l/titer ± SE)

SWZ107^a	Switzerland	1980	4: P1.2	104	Positive	≧64	28 ± 4
NG6/88^a	Norway	1988	NT: P1.1	173	Positive	4	115 ± 23
CU385^b	Cuba	1980	4: P1.15	5	Positive	4	116 ± 1
IH5341	Finland	1985	15: P1.7, 16	ND	Positive	16	176 ± 61
BZ198	Netherlands	1986	NT: P-	154	Positive	≧64	362 ± 1
NMB	US	1968	2b: P1.2, 5	ND	Positive	16	244 ± 20
8047	US	1978	2b: P1.2	ND	Positive	16	1125 ± 50
H44/76	Norway	1976	15: P1.7, 16	5	Positive	24	99 ± 16
1000^a	USSR	1989	NT: P1.5	61	Positive	<4	287 ± 12
S3446^b	US	1972	14: P1.23, 14	11 (A1 cluster)	Positive	<4 (static = 16)	585 ± 151
H355^b	Norway	1973	15: P1.15	11 cluster	Positive	<4 (static = 4)	656 ± 141
BZ232^a	Netherlands	1964	NT: P1.2	76	Negative	<4	1493 ± 18
NG3/88^a	Norway	1988	8: P1.1	A4 cluster	Negative	<4	498 ± 105
MC58	UK	1985	15: P1.7, 16b	5	Negative	<4	627 ± 121
M136^b	US	1968	11: P1.15	D1 cluster	Negative	<4	1056 ± 81
M986^b	US	1963	2a: P1.5, 2	B2 cluster	Negative	<4	1442 ± 206
NGP165	Norway	1974	NT: P1.2	37	Negative	<4	138 ± 6

^aDenotes strains that have been characterized further by multilocus sequence typing [Maiden, 1998].
^bDenotes strains obtained from the Frasch collection, US FDA. 8047 was obtained from W. Zollinger, Walter Reed Army Institute of Research, Washington, D.C. MC58 is the strain selected by TIGR for genomic sequencing. J351 was obtained from M. Sarvas, National Public Health Institute, Helsinki, Finland.
# The remaining strains are from the collection described by Seiler et al. [Seiler, 1996]. ET data are from Caugnant et al. [j. Infect. Dis. (1990) 162: 867-874], and Seiler et al.
^cBy indirect fluorescence flow cytometry with anti-rNspA antisera.
^dDilution of anti-rNspA antisera that when incubated for 60 min. with bacterial cells and 20% human complement yielded ≧50% decrease in CFU/ml, compared to that at time 0. “Static” refers to strains that were inhibited but not killed in the assay (≧50% but <100% survival at 60 mins).
^eTitre defined as dilution of capsule extract giving 50% inhibition of antibody binding to meningococcal B polysaccharide antigen in an ELISA.

Claims

1. A composition comprising a first isolated biological molecule from a Neisseria bacterium and a second isolated biological molecule from a Neisseria bacterium, wherein the first biological molecule is from ORF 287 and the second biological molecule is from ORF 961.

2. The composition of claim 1, wherein said first biological molecule and said second biological molecule are from the same Neisseria species.

3. The composition of claim 1, wherein said first biological molecule and said second biological molecule are from different Neisseria species.

4. The composition of claim 1, wherein said first biological molecule and said second biological molecule are from N.meningitidis and/or N.gonorrhoeae

5. The composition of claim 1, wherein said first biological molecule and said second biological molecule are from N.meningitidis.

6. The composition of claim 1, wherein said first biological molecule comprises an amino acid sequence with at least 65% sequence identity to a contiguous sequence of amino acids selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and an immunogenic fragment comprising at least 10 contiguous amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; and said second biological molecule comprises an amino acid sequence with at least 65% sequence identity to a contiguous sequence of amino acids selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and an immunogenic fragment comprising at least 10 contiguous amino acids of SEQ ID NO:5 or SEQ ID NO:6:

7. The composition of claim 6, wherein said first biological molecule is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

8. The composition of claim 7, wherein said first biological molecule is SEQ ID NO:1.

9. The composition of claim 6, wherein said second biological molecule is selected from the group consisting of SEQ ID NO:5, and SEQ ID NO:6.

10. The composition of claim 9, wherein said second biological molecule is SEQ ID NO:6.

11. A composition comprising a first isolated biological molecule from a Neisseria bacterium and a second isolated biological molecule from a Neisseria bacterium, wherein

(a) said first biological molecule comprises an amino acid sequence with at least 65% identity to the contiguous sequence of amino acids of SEQ ID NO:1 or an immunogenic fragment comprising at least 10 contiguous amino acids of SEQ ID NO:1; and

(b) said second biological molecule comprises an amino acid sequence with at least 65% identity to the contiguous sequence of amino acids of SEQ ID NO:6 or an immunogenic fragment comprising at least 10 contiguous amino acids of SEQ ID NO:6.

12. The composition of claim 11, wherein said first biological molecule comprises the amino acid sequence of SEQ ID NO:1, and said second biological molecule comprises the amino acid sequence of SEQ ID NO:6.

13. The composition of claim 1, further comprising an adjuvant.

14. The composition of claim 12, further comprising an adjuvant.