Haemophilus influenzae b (Hib)

Our group is responsible for batch release testing the different Hib vaccines and for establishing related standards and reference reagents. We also carry out a diverse programme of collaborative research and development. 

Haemophilus influenzae is a gram-negative bacterium which can exist in two forms – encapsulated (serotypes a-f, based on the type of capsular polysaccharide) and non-encapsulated (non-typeable).

The non-typeable forms can cause non-invasive respiratory tract infections and otitis media – middle ear infection.

The encapsulated forms can cause more serious invasive conditions including bacteraemia, pneumonia and meningitis – almost exclusively in children under 5. It is transmitted through the respiratory tract from infected to susceptible people.

Haemophilus influenzae type b (Hib) is the most virulent strain of the encapsulated form. Current vaccines against Hib are made from capsular polysaccharide conjugated to a carrier protein to make them immunogenic in the infants. Hib conjugate vaccine is an important vaccine in the infant immunisation programme and it has reduced Hib disease dramatically worldwide.

Standards

The Hib group is responsible for preparing and maintaining reference standards and reagents used in the production and control of Hib vaccines, epidemiological and diagnostic investigations and vaccine clinical trials as well as various R&D studies. These include:

• the international standard for Hib polysaccharide
• Hib oligosaccharide-conjugated to human serum albumin (HbO-HA)
• human anti-Hib reference serum

Control testing

Hib conjugate vaccines are made from the Hib capsular polysaccharide known as PRP conjugated to a carrier protein such as tetanus toxoid (TT), non-toxic, cross-reacting material (CRM) or outer membrane proteins (OMP).

The vaccine is available in monovalent presentation, combined with meningococcal C conjugate vaccine (Hib/MenC) or with diphtheria, tetanus and whole cell/acellular pertussis-based vaccines, with/without injectable polio (IPV) and/or hepatitis B (HepB) vaccines.

The Hib Group carries out scientific testing of some Hib-containing vaccine formulations as part of NIBSCs role as a National Control Laboratory.

A number of tests are used by our group to ensure the safety, efficacy and consistency of the Hib vaccine batches used in the infant primary and booster immunisation programmes. The group also supports the World Health Organisation (WHO) by testing various Hib vaccines for the WHO Prequalification Programme.

As well as routine testing of Hib vaccines, we invest a great deal of effort into developing new assays to improve quality control testing of Hib vaccines (see R&D section).

We carry out these tests as part of batch release of Hib vaccines:

• appearance
• identity test for the polysaccharide
• identity test for the carrier protein
• total and free polysaccharide content
• molecular size
• endotoxin content
• manufacturer’s protocol review

Research and development

Our group’s work is supported and complemented by a number of R&D projects.

Development of new assays for Hib testing

Quality control testing of Hib conjugate vaccines relies almost completely on physico-chemical methods to monitor consistency of production and identify any trends in batches over time. Biological testing is carried out only to ensure safety.

The tests for total and free polysaccharide content are considered surrogate potency tests and are usually carried out using the high-performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) assay. However, for some combination vaccines such as Pediacel, this assay has been problematic because of interference of the vaccine matrix with the assay.

We have developed and validated an alternative, ELISA-based assay for use on final fills of Hib-containing combination vaccines. The ELISA assay is based on using a set of anti-PRP antibodies for capturing and detecting PRP in Hib vaccines. The test was accredited to 17025 Standard in 2012 and is now used for batch release testing.

Relationship between copy number of the capsulation gene and bacteria virulence

Despite the effectiveness of Hib conjugate vaccines, there was an increase in invasive Hib disease in immunised children in the UK between 1999 and 2003.

A study to determine the number of copies of the capsule genes (capb) in children from the

vaccine failure group showed that many strains contained multiple copies of the capb locus compared with strains from unvaccinated children.

Our group is investigating the expression of capsular polysaccharide in those Hib isolates with multiple copies of the capb locus and their susceptibility to antibody and complement-mediated killing/binding assays in a collaboration with Public Health England.

Novel new generation Hib vaccines 

Producing conjugate vaccines is an expensive and complicated chemical process, involving covalently coupling the capsular polysaccharide to the carrier protein. This limits their use in poor countries.

In a collaboration with XStalBio and University College London, we are investigating the possibility of using Xstalbio proprietary technology to develop a cheap and novel self-assembled polysaccharide-carrier protein formulation to act as virtual conjugate vaccines. 

Pre-clinical evaluation of vaccine in development stages

As well as our work on Hib, the group is also actively involved in other R&D projects on vaccines that are still being developed, such as those against group B streptococcus diseases and otitis media caused by non-typeable Haemophilus influenzae.

Nanotechnology and skin immunisation

In line with our role in improving current vaccines, the Hib group is actively involved in a collaboration with the University of Hertfordshire to evaluate nanotechnology as a platform to improve the delivery of vaccines transcutaneously.