Evaluation of the immunogenic property of NT H. influenzae protein D with Neisseria meningitidis OMV in BALB/c.

Ava Behrouzi,Mehdi Davari,Saeid Bouzari,Mana Oloomi,Hoora Mazaheri,Seyed Davar Siadat

doi:10.3855/jidc.7513

Ava Behrouzi, Mehdi Davari + Show 4 more

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https://doi.org/10.3855/jidc.7513

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Abstract

Identifying ideal non typeable Haemophilus influenzae (NTHi) vaccine candidates has not been easy due to extensive sequence and antigenic variation among gene products interacting with the immune system. Protein D (PD) is a highly conserved 42 kDa surface lipoprotein available in all H. influenzae, including NTHi. In this study, the gene encoding PD was cloned from H. influenzae and expressed in Escheriachia coli TOPO10 cell in pBAD vector. Arabinose was used to express recombinant protein. In order to purify the protein, Ni-NTA agarose was used to perform affinity chromatography. Purified PD and PD mixed with outer membrane vesicle (OMV) and alum adjuvant were used for subcutaneous immunization in BALB/c mice. After vaccination, IgG responses to PD-OMV, PD-alum, and PD alone were determined by enzyme-linked immunosorbent assay (ELISA). The recombinant PD containing His6 residues showed a molecular weight of 42 kDa. Anti-PD IgG was detected after first immunization in all groups of mice compared to the negative control group, and it increased after first vaccination, but results showed that the addition of OMV to PD led to a remarkable increase in IgG responses. Our results suggest an important role for OMV as an adjuvant and show how it could potentially be used when conjugated to H. influenzae PD or other safe subunit vaccine candidates.

Highlights

Identifying ideal non typeable Haemophilus influenzae (NTHi) vaccine candidates has not been easy due to extensive sequence and antigenic variation among gene products interacting with the immune system
The amplified hpd polymerase chain reaction (PCR) product (1,095 bp) was cloned into digested pBAD and the generated plasmid was moved into E. coli TOPO10 cells by transformation
It should be noted that IgG1 response was statistically higher than IgG2a (Figure 3). These results showed that addition of outer membrane vesicle (OMV) to Protein D (PD) remarkably increased the IgG responses, and indicated that OMV can act as a potent adjuvant to enhance the antibody responses

Summary

Introduction

Identifying ideal non typeable Haemophilus influenzae (NTHi) vaccine candidates has not been easy due to extensive sequence and antigenic variation among gene products interacting with the immune system. Purified PD and PD mixed with outer membrane vesicle (OMV) and alum adjuvant were used for subcutaneous immunization in BALB/c mice. Vaccines against typeable Haemophilus influenzae strains are being used extensively, but they do not protect against infections caused by NTHi strains [3]. To produce a vaccine against Haemophilus influenzae infections, several H. influenzae proteins have been studied [7,8]. The identification of NTHi vaccine candidates is not simple, because NTHi display extensive variations among different genes interacting with the immune system [3,9,10]. One promising antigen is the outer membrane protein D (PD) [11]

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