Title:  OMP26 antigen from Haemophilus influenzae

United States Patent:  6,245,338

Assignee:  Provalis UK Limited (GB)

Filed:  December 22, 1997

Foreign Application Priority Data:  Jun 27, 1995[GB] (9513074)

A novel antigenic protein derived from the outer membrane of H. influenzae is provided. DNA sequences encoding such a protein are also provided as are vaccines comprising the protein and methods of immunizing a subject against H. influenzae infection. The invention also includes methods for the prophylaxis or treatment of respiratory tract infections or otitis media, as well as methods for the detection of H. influenzae, and kits for use in such methods.

Description of the Invention

The present invention relates to a novel antigen of Haemophilus influenzae, vaccines comprising it and its use in therapy and diagnosis.

H. influenzae is a Gram-negative aerobic heterotrophic bacteria with the form of rods (Krieg and Holt (ed), Bergey's Manual of Systemic Bacteriology, pp 563 (1984). It is a pathogen in acute respiratory infections and is also found in patients with chronic bronchitis and otitis media.

We have now identified, and purified, a unique 26 kDa outer membrane protein (called OMP26) from NTHI, and have surprisingly found that this protein can, when used as an immunogen, induce protective immune responses against infection with homologous and heterologous strains of NTHi. This protein has a molecular mass on SDS-PAGE similar to P5, but has been found to be distinctly different from the protein.

The outer membrane protein P5 is one of two lower molecular mass bands on SDS-PAGE gels used to subtype H. influenzae strains, and has an apparent molecular mass of 25-27 kDa. The P5 protein is heat-modifiable, demonstrating an apparent mass of 35 kDa after heating for 30 min at 100.degree. C. in the presence of .beta.-mercaptoethanol. Recently, another protein expressed by NTHi, called a fimbrin protein, has been characterised and shown to have similar molecular mass properties, heat modifiability and a 92% sequence homology to the previously described P5. The protein, OMP26, does not demonstrate either sequence homology or heat-modifiable characteristics as defined for either P5 or the fimbrin protein.

Thus, in a first aspect, the present invention provides a protein having a molecular weight of 26 kDa, as determined by SDS-PAGE, which protein is an outer membrane protein of H. influenzae. This protein is designated OMP26.

In particular, the protein of the invention has the amino acid sequence shown in FIG. 1, or one substantially homologous thereto. In a separate embodiment, the protein of the invention has the amino acid sequence shown in FIG. 1 commencing from amino acid no. 24, or one substantially homologous thereto. The first 23 amino acids constitute a "signal" sequence and it will be appreciated that a protein minus this sequence will be equally applicable. The protein of the invention is an immunogen and is thus capable of inducing an immune response which will protect against infection with H. influenzae.

In the context of the present invention proteins which are "substantially homologous" to OMP26 may be 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 99% homologous. Preferably, the protein will be at least 70% homologous, more preferably 80% homologous, even more preferably 90% homologous and most preferably 95% homologous. The skilled man will appreciate that the percentage degree of homology is one factor only. What is important is that the protein retains its antigenic effect. Thus, it is reasonable to have a protein having a relatively low degree of homology, for instance 40%, while retaining the antigenic activity discussed herein.

In addition, it is known in the art that "conservative" or indeed "semi-conservative" changes can be made to the amino acid sequence of a protein which will not alter its fundamental activity. For example, amino acids such as glycine, valine, leucine and isoleucine, which all have aliphatic side chains, may often be substituted for each other without substantially altering the biological activity of the protein. Similarly, amino acids such as phenylalanine, tyrosine and tryptophan, which all have aromatic side chains, may be substituted for each other. Such proteins which retain the antigenic effect described herein are within the scope of the present invention.

It is also possible that antigenic parts or regions of OMP26 can be employed to induce the protective effect against H. influenzae. Such antigenic parts or regions are also within the scope of the present invention.

In a second aspect, the present invention provides a nucleic acid sequence, preferably DNA, which codes for a protein of the invention, variants thereof as described above or indeed antigenic parts or regions. In particular, the invention provides a DNA sequence as shown in FIG. 1 which codes for OMP26. The skilled man will appreciate that due to the degeneracy of the genetic code it is possible to make conservative changes to the DNA sequence which will not result in changes to the amino acid sequence of the protein. Thus, such DNA sequences are also within the scope of the present invention. Suitably, nucleic acid of the invention can form part of a vector such as a plasmid.

As discussed herein, the proteins of the invention stimulate an immune response against H. influenzae and thus, in a third aspect, the present invention provides a vaccine formulation comprising a protein of the invention, as defined herein, optionally together with one or more carriers and/or adjuvants.

In a fourth aspect, the invention provides the use of the protein of the invention, as defined herein, in the preparation of a vaccine against H. influenzae.

The vaccine composition of the invention can be used to immunize a subject against H. influenzae infection. Therefore, the invention provides, in a fifth aspect, a method of immunizing a subject against infection by H. Influenzae, which comprises administering to the subject a vaccine composition of the invention. The vaccine compositions of the invention can be used to produce both systemic immunity and/or mucosal immunity.

In a sixth aspect, the present invention provides a method for the prophylaxis or treatment of respiratory tract infections or otitis media which comprises the step of administering to a subject a vaccine composition of the invention.

In other aspects the invention provides:

(a) The use of a protein of the invention, as defined herein, in the diagnosis of H. Influenzae infection;and

(b) A kit for use in the diagnosis of H. Influenzae infection comprising a protein of the invention, as defined herein.

Preferred features of each aspect of the invention are equally preferred for each other aspect mutatis mutandis.

Claim 1 of 31 Claims

What is claimed:

1. A vaccine formulation comprising an isolated protein which (i) is an outer membrane protein of Haemophilus influenzae or an immunogenic fragment thereof and (ii) comprises the amino acid sequence as set forth in SEQ ID NO.2, an immunogenic fragment of said sequence, a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO.2 and having an immunogenic epitope of SEQ ID NO:2 or an immunogenic fragment of a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO.2 wherein said fragment has an immunogenic epitope of SEQ ID NO:2, together with one or more carriers and/or adjuvants.

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