Immunogenicity and protective immunity against otitis media caused by pneumococcus in mice of Hib conjugate vaccine with PsaA protein carrier

Abstract

This study evaluated the immunogenicity and protective immunity of a Hemophilus influenzae b (Hib) polysaccharide conjugate vaccine with the pneumococcal surface adhesin A (PsaA) protein carrier in young mice. The Hib polysaccharide was conjugated with the rPsaA protein carrier, which was produced using recombinant DNA technology. A total of 15 young mice aged 3 weeks to 5 weeks were immunized with the conjugate vaccine, and another 15 young mice of the same age were immunized with the licensed Hib-tetanus toxoid (TT) vaccine. Furthermore, the third group of 15 young mice was inoculated with phosphate buffer saline as control. The immunized mice were inoculated with pneumococcus in the middle ear. Results showed that IgG antibody responses against both the PsaA protein and Hib polysaccharide were observed in the Hib-PsaA group. However, no statistical difference was observed in the titer of IgG against the Hib polysaccharide between Hib-PsaA and Hib-TT groups. The elimination rate of pneumococcus and the inflammation of the middle ear showed the effectiveness of protective immunity against otitis media caused by pneumococcus. Our results suggest that the Hib polysaccharide can be successfully conjugated with rPsaA via amide condensation. This new Hib-PsaA conjugate vaccine can induce both anti-PsaA and anti-Hib immune responses in young mice and elicit effective protection against acute otitis media caused by pneumococcus.

Keywords

conjugate vaccine / pneumococcal surface adhesin A / Hemophilus influenzae b / immunogenicity / otitis media

Introduction

Acute otitis media (AOM) is one of the most common infectious diseases in the world, particularly in children. Otitis media (OM) and its complications represent the main causes of hearing loss, which are usually more severe in developing countries than in other countries. Furthermore, OM is one of the most common diseases treated by antibiotics. However, in recent years, the resistance of bacteria to antibiotics has significantly increased. With the goals of decreasing antibiotic usage and reducing AOM treatment resistance, the development of vaccines against AOM holds significant promise for reducing cost and preventing the hearing loss caused by OM.

Pneumococcal conjugate vaccines (PCV), such as the seven-valent PCV (PCV7) and Hemophilus influenzae b (Hib)–tetanus toxoid (TT) conjugate vaccine, have been in development for several years. The effectiveness of PCV in reducing the risk of AOM in both infants and young children has been reported [ 1]. However, the effectiveness of PCV is limited by the serotypes included in the vaccine. The Hib-TT vaccine showed good immunogenicity and could potentially reduce the burden of AOM [ 2, 3]. Given that pneumococcus and H. influenzae are the most common bacteria that cause AOM [ 4], the development of a conjugate vaccine against these two bacteria would help prevent this infection. The most common bacteria isolated from the effusion fluid in the middle ears of patients with OM are pneumococcus and H. influenzae, which are also found in AOM [ 5]. If a conjugate vaccine could induce immune responses against these two bacteria, such a vaccine would play an important role in AOM prevention. However, existing vaccines can only induce an immune response against a bacterium. Moreover, components of TT may disturb the immune program in children vaccinated with other TT-based vaccines [ 6, 7].

This study investigated the ability of a conjugate vaccine to induce immune responses against pneumococcus and H. influenzae. The proposed vaccine was produced by conjugating the Hib polysaccharide with pneumococcal surface adhesin A (PsaA) as a protein carrier. The aim of our investigation is to provide experimental evidence for an improved AOM vaccination strategy.

Materials and methods

Laboratory animals

A total of 45 SPF-grade female BALB/c mice aged 3 weeks to 5 weeks with weights between 11 and 16 g were obtained and housed in an SPF-grade experiment room at the Public Health Clinic Center of Fudan University, China.

Bacterial strains, vector, and antigen

Type 14 Streptococcus pneumonia bacteria were obtained from the National Center for Medical Culture Collections of China. pET-28a (vector), Escherichia coli DH5a, and E. coli BL21 (DE3) (host bacteria) were obtained from Haigui Bio-Tech Co. Ltd., Shanghai, China. The antigen for Hib polysaccharide and the licensed conjugate vaccine for Hib-TT were obtained from Wuhan Institute of Biological Products, China.

Main equipment and reagents

The restriction enzymes, NcoI and Hind III, T4 DNA ligase, Taq DNA polymerase, protein markers, and DNA markers were obtained from Takara Co. Ltd., Japan. Agarose gel was obtained from Amersham Biosciences Co., USA. The plasmid extraction kit and the rubber recycling kit were purchased from Axygen Co. Ltd. Bovine serum albumin (BSA), sheep anti-mouse IgG conjugated with horseradish peroxidase (HRP), ADH, and EDTA were purchased from Sigma-Aldrich Corp., USA. The CNBr was obtained from J&K Scientific Ltd. The ELISA plate-washing machine and microplate reader were obtained from Bio-Rad Co. Ltd. The automatic potentiometric titration machine was obtained from Metrohm Co. Ltd., Switzerland. The AKTA chromatography system was obtained from GE Co. Ltd., USA.

The PCR amplification of PsaA gene

The gene amplification of PsaA was based on the gene sequence of PsaA in GenBank. The upstream primer and downstream primer were designed as follows: upstream primer 5′-CATGCCATGGCTGCTAGCGGAAAAAAAGAT-3′, with the underlined sequence showing the restriction site of NcoI; downstream primer 5′-CGCAAGCTT TTATTTTGCCAATCC TTCAG-3′, with the underlined sequence showing the restriction site of HindIII. The primers were synthesized by Shanghai Sangon Biotech Co. Ltd. The PsaA gene was amplified from the pneumococcal chromosomal DNA as the template with PCR. The PCR conditions were 35 cycles of 94 °C for 3 min, 94 °C for 30 s, 48 °C for 30 s, and 72 °C for 30 s. At the end of these cycles, an extension step was performed at 72 °C for 10 min. The PCR products were identified via agarose gel electrophoresis.

Construction, selection, and identification of the expression vector

The PsaA gene and pET-28a vector were enzyme-digested by the restriction enzymes HindIII and NcoI, respectively. After enzyme digestion, the pET-28a vector and the target gene PsaA were retrieved and linked via the T4 DNA ligase reaction at 22 °C. The vector containing the PsaA gene was transferred to the competent cell line E. coli DH5a, which was then cultured in LB culture media containing kanamycin (50 mg/ml). Positive clones were selected and identified by enzyme digestion, and the correct clone was confirmed via gene sequencing by Shanghai Sangon Biotech Co. Ltd.

Expression of the PsaA gene and purification of the PsaA protein

The recombinant expression clone pET-28a-PsaA was transferred to the competent cell line E. coli BL21 (DE3). The bacteria were cultured in LB liquid culture media containing kanamycin (50 mg/ml) at 37 °C for 2 h and then harvested at an A value of 0.6–0.8. IPTG was then added to induce PsaA protein expression for 4 h at a final concentration of 1 mmol/L. The bacteria solution was centrifuged, resuspended, and then disrupted by ultrasound. After centrifugation, the supernatant fraction was collected and applied on the Ni-NTA agarose column (His tag affinity column) to purify recombinant protein. The target protein was eluted from the resin with 25 mmol/L Tris buffer containing 0, 20, and 200 mmol/L imidazole. The eluent was collected and the PsaA protein was identified by 12% SDS-PAGE.

Preparation of the Hib polysaccharide-PsaA conjugate

The conjugate was produced by the amide condensation method. A total of 10 mg/ml of Hib polysaccharide was activated by CNBr. The activated Hib-ADH was then conjugated with PsaA at a weight ratio of 1:1 by using ADH and EDAC as connecting agents. After purification, the conjugate was monitored by spectrophotometry at the wavelengths of 206 and 280 nm. After aseptic filtration, the Hib-PsaA conjugate was obtained. The polysaccharide and protein contents and the relative molecular mass were determined.

Quantitative determination of the protein and polysaccharide content of the conjugate

The modified Lowry method was used to determine the protein concentration. The ammonium molybdate method was used to determine the phosphor content. The polysaccharide content of the conjugate was determined by a modified ammonium molybdate method.

Immunogenicity detection for the PsaA-Hib conjugate vaccine

A total of 45 infant BALB/c mice with ages of 3 weeks to 5 weeks were randomly divided into 3 groups with 15 mice in each group. Hib-PsaA conjugate vaccine, the licensed Hib-TT conjugate vaccine, and phosphate buffer saline (PBS) as a control were inoculated by intraperitoneal injection. The Hib polysaccharide concentration in the 2 types of conjugate vaccines was 5 mg/ml. The mice were inoculated with 0.5 ml of vaccine or PBS once every 2 weeks for a total of 3 injections.

Detection of antibody responses for anti-PsaA IgG and anti-Hib polysaccharide IgG

Two weeks after the last immunization, the blood of each mouse was collected for antibody detection. Titers of anti-Hib polysaccharide IgG and anti-PsaA IgG in the serum of each group of mice were determined by ELISA. The ELISA plates were coated with approximately 9.7 mg/ml (BSA concentration) Hib-BSA conjugate solution from Wuhan Institute of Biological Products, China, and approximately 9.9 mg/ml of PsaA solution. The plates were then washed, blocked, and rewashed. The serum samples from each group of mice were diluted to 1:50 and then double-diluted. The negative control sample was diluted with the same method. A total of 100 µl of each sample was added to the coated plates and then incubated at 37 °C for 60 min. After washing, sheep anti-mouse IgG-HRP was added to the plates and then incubated at 37 °C for 30 min. After the development of the color reaction, the optical density (OD) value was determined by a microplate reader.

Pneumococcal testing to the middle ear of mice

The pneumococcal trial was performed in an ABSL-2 laboratory. According to Sabirov et al. [ 8], 10 µl of type 14 pneumococcus, including 5.6×10⁶ cfu, was injected into the middle ear of the mice for testing. Two weeks after the last immunization, the mice in Hib-PsaA and PBS groups were tested by being injected with pneumococcus in the middle ear. Three days after the pneumococcus injection, 7 mice in Hib-PsaA and PBS groups were sacrificed, and their acoustic vesicles were exposed. The middle ears were lavaged with 50 µl saline (5 µl × 10 times) through the tympanic membrane. The lavage fluid was double-diluted, smeared onto blood plates, and then placed into a 37 °C 5% CO₂ incubator. After 24 hours, pneumococci were identified according to their morphology and the Optochin test; the amount of bacteria was also counted. The acoustic vesicles of the 2 groups of mice were removed. Following 7 mice in each group being sacrificed, the remaining 8 mice in each group were sacrificed 7 days after injection. The vesicles were fixed with 4% paraformaldehyde and then decalcified with 10% EDTA for 1 week. Frozen sections were made, stained with hematoxylin and eosin, and then analyzed histopathologically.

Results

Identification of the PsaA gene amplification product

The PsaA gene product amplified via PCR was identified via agarose gel electrophoresis. The 870-bp DNA band was confirmed to be the PsaA gene fragment (Fig. 1).

rPsaA protein expression and purification

The rPsaA protein was expressed by E. coli BL21 (DE3), which was transformed with a pET-28a plasmid carrying the PsaA gene. The purity of this protein was over 90%, as shown by 12% SDS-PAGE (Fig. 2).

The PsaA protein was conjugated with the Hib polysaccharide via amide condensation. The conjugation results showed that the peak OD value of the collecting liquid for PsaA chromatographed by gel column Sepharose 4FF was approximately 145 ml (Fig. 3); whereas that of the conjugate formulation was approximately 58 ml (Fig. 4). Given that the relative molecular mass of the conjugate was larger than the protein, the position of the peak OD value in the chart was moved forward, thus confirming effective conjugation. The contents of PsaA and Hib polysaccharide in the conjugate were 42 and 31 mg/ml, respectively. The free polysaccharide level was 15.2%. The ratio of polysaccharide to protein in the conjugate was 0.74:1.

Serum antibody titers of anti-PsaA IgG and anti-Hib polysaccharide IgG in the mice immunized with the conjugate vaccine

The results of the IgG titers for each group of mice immunized with the Hib-PsaA or the Hib-TT conjugate vaccine are shown in Table 1 and Fig. 5. The geometric mean titers (GMTs) of mice anti-PsaA IgG in the Hib-PsaA and PBS groups were 68.42±15.55 and 8.71±2.09, respectively. A statistically significant difference was observed between the two groups, as determined by a two-sample t-test (t = 14.74, P<0.001). The GMTs of the anti-Hib polysaccharide IgG of Hib-PsaA, Hib-TT, and PBS group were 51.64±16.91, 49.29±15.93, and 8.52±2.10, respectively. According to a two-sample t-test (t = 0.391, P = 0.699>0.05), no significant difference was observed in the GMT of anti-Hib polysaccharide IgG between the mice immunized with the Hib-PsaA vaccine and those immunized with the Hib-TT vaccine. However, a significant difference in the GMT of anti-Hib polysaccharide IgG was observed between the Hib-PsaA and PBS groups (t = 9.80, P<0.001), and the Hib-TT and PBS groups (t = 9.83, P<0.001), as determined by a two-sample t-test. Our results also showed that the Hib-PsaA vaccine produced in this study induced similar immunogenicity against the Hib polysaccharide as the Hib-TT vaccine.

However, the Hib-PsaA vaccine induced a strong immunologic response against PsaA with an IgG GMT of 68.42. This result showed the advantage of using PsaA as a protein carrier compared with using Hib-TT vaccine alone. Therefore, our results showed that the Hib-PsaA vaccine could induce immunologic responses against two antigens: PsaA and the Hib polysaccharide.

Results of the pneumococcus testing

Three days after pneumococcus injection into the middle ear, the average number of bacteria in the lavage fluid of the middle ear of mice in the Hib-PsaA group was 1.84×10⁴. For the PBS group, this value was 2.34×10⁵. A significant difference was observed between these two groups by a two-sample t-test (t = 2.57, P<0.05).

The inflammatory reactions in the ear of the mice in the Hib-PsaA group and PBS group were modest and strong, respectively, three days following the challenge with pneumococcus (Fig. 6A and 6B). In the Hib-PsaA group, few leukocytes infiltrated the middle ear, and the cells of the tympanic tissue were slightly swollen. By contrast, in the PBS group, many leukocytes infiltrated the middle ear, and the cells of the tissue were swollen and significantly degenerated. Similar results were found seven days after the challenge with pneumococcus. The inflammation in the middle ears of the mice in the PBS group was even stronger than in the Hib-PsaA group, in that the swelling of cells was more severe; whereas, the change was unremarkable in the Hib-PsaA group (Fig. 6C and 6D).

Three days after the challenge, the tympanic membranes in the PBS group were thicker and more swollen than those in the Hib-PsaA group. The overall level of inflammation was also higher in the PBS group than in the Hib-PsaA group (Fig. 7A and 7B). On the seventh day post-test, the tympanic membranes in the Hib-PsaA group were still intact without obvious swelling, whereas those in the PBS group were broken and clearly swollen (Fig. 7C and 7D).

Discussion

AOM is one of the most common infectious diseases in children, and OM and its complications are major causes of hearing loss. Notably, these problems are more severe and widespread in developing countries than in other countries. Approximately 70% to 90% of children younger than six years of age have experienced at least one episode of OM [ 9], and more than 75% of children younger than three years of age have experienced OM [ 10]. OM is commonly treated with antibiotics. However, because of the overuse and abuse of antibiotics in recent years, drug-resistant bacterial strains have been increasing. Therefore, antibiotic resistance is a growing problem. Vaccination against OM may decrease antibiotic use, overcome the effect of antibiotic resistance, decrease the financial burden on the families of children with AOM, and prevent children from suffering hearing loss caused by AOM [ 11].

Given that a polysaccharide vaccine does not induce a T cell-dependent immune response, it is only effective in adults and children above 2 years of age. The Hib-PsaA conjugate vaccine, which can induce a T cell-dependent immune response, will be effective in children under 2 years of age who suffer the highest rates of pneumococcal disease. To test the Hib-PsaA conjugate vaccine, young mice were chosen in this study. BALB/c mice aged 3 weeks to 5 weeks can still be used to test T cell-dependent immune response because these mice reach adulthood after 60 days.

Recently, marketed vaccines available both locally and abroad include the PncT and the Hib-TT conjugate vaccines. Currently, a conjugate vaccine for the pneumococcal polysaccharide–protein D of H. influenzae of an uncertain type is being studied abroad. Pneumococcus, H. influenza, and Moraxella catarrhails are the most common types of bacteria that cause AOM and account for 40%–50%, 20%–30%, and 10%–15% of cases, respectively [ 12]. The most common bacteria isolated from the middle ear effusion of secretory OM cases are also pneumococcus and H. influenza [ 5]. Therefore, the study and use of vaccines against these bacteria will be significant in the prevention of AOM and secretory OM. Previous studies have demonstrated that inoculation with a 7-valent pneumococcal conjugate vaccine could significantly decrease parasitism by pneumococcus of related vaccine serotypes in the nasopharynx [ 13]. The Hib-PsaA conjugate vaccine combines antigens from these two types of bacteria. Moreover, this conjugate vaccine avoids interference with vaccine antigens, including TT, in planned vaccines, such as the pertussis–diphtheria–tetanus triple vaccine (DPT) [ 6]. Additionally, as a polysaccharide–protein conjugate vaccine, our formation may also be effective in children younger than two years of age, who represent a high-risk population for AOM.

The capsular polysaccharide of pneumococcus has more than 90 different serotypes. Therefore, the antibodies induced by the inoculation of vaccines against the capsular polysaccharide antigens of pneumococcus show type specificity, i.e., the vaccines can provide serotype-specific immune protection against pneumococcal infections [ 14]. Although the developed 7-valent pneumococcal polysaccharide–protein conjugate vaccine includes 7 major pathogenic serotypes, the major serotypes among countries and districts are different, and the pathogenic serotype in the same district can change over time [ 15, 16]. Therefore, the vaccine produced from the pneumococcal polysaccharide antigen has limited immune cross-protection. PsaA, PspA, and Ply, as the major pneumococcus protein antigens, possess antigenicity and provide immune cross-protection from different pneumococcal polysaccharide serotypes [ 17]. PsaA is a surface lipoprotein that plays a key role in the adhesion of pneumococcus to the respiratory mucosa. PsaA is a common surface protein antigen of various types of pneumococcus that shows conservative hereditary, species specificity, and good antigenicity related to toxicity. Since the first report on PsaA in 1990, studies on the PsaA protein has become increasingly common. In particular, studies have found that PsaA is a good antigen protein to decrease parasitism by pneumococcus in the human body [ 18– 20]. In the current study, our Hib polysaccharide conjugate vaccine used PsaA as a protein carrier and may confer better immune protection than conjugate vaccines by using other proteins as carriers. Given the polymorphology of the PsaA family, PsaA may have limited immunogenicity, thus suggesting that PsaA may not be identical across serotypes of pneumococcus. Furthermore, this situation may limit the cross-protection provided by the vaccine. This hypothesis will be further explored by our laboratory in the future.

The results of this study showed that the PsaA protein can be successfully produced on a large scale by genetic engineering through recombined PsaA gene amplification and transformation of bacteria with plasmids. The expressed and purified recombined PsaA protein showed high output and purity and satisfied our experimental parameters. Given the limited time frame of this study, the conjugation and optimization methods still require further exploration. In particular, the polysaccharide–protein ratio, conjugation conditions, and purification methods will be further optimized in future experiments.

Following vaccination of young mice, the GMT of anti-PsaA IgG in the Hib-PsaA conjugate vaccine group was 68.42. An ideal antibody titer was acquired, thus indicating that the conjugate showed antigenicity against pneumococcus. Our results also indicated that the GMT of the anti-Hib polysaccharide IgG antibody elicited no significant differences between the Hib-PsaA and the Hib-TT conjugate vaccine groups (P>0.05), thus indicating that the Hib-PsaA conjugate vaccine against Hib used in this study showed equivalent antigenicity compared with the available Hib polysaccharide conjugate vaccine.

The serotypes most commonly associated with infection of pneumococcus are 14, 6B, 19F, 18C, 23F, 4, and 9V [ 21]; however, 19F, 3, 6B, 14, 19A, and 23F are the leading serotypes in children with AOM [ 22]. Therefore, serotype 14 pneumococcus was chosen for middle ear testing. The bacterial test results indicated that the bacterial colonies cultured in the lavage fluid from the middle ear on the third and seventh days after pneumococcus injection in the experimental groups were statistically lower than those in the control group (P<0.05). Furthermore, the extent of the middle ear inflammatory response in the experimental group was lower than the control group on the third day of the histopathological study. Moreover, the differences in the extent of the inflammatory response between the experimental and control groups were significant on the seventh day. These results indicated that inoculation of the vaccine could effectively protect young mice against the inflammatory response after pneumococcus infection.

The results of this study highlight the following advantages of the Hib-PsaA conjugate vaccine. First, as a polysaccharide–protein conjugate vaccine, the Hib-PsaA may induce an effective immune response in infants. Second, the PsaA used in this vaccine was a pneumococcal protein antigen and may demonstrate cross-immune protection in various serotypes of pneumococcus. Third, this vaccine induced an IgG antibody response against PsaA and the Hib polysaccharide; this finding is important because PsaA and Hib were formulated from components of the dominant pathogens in AOM. As a result, this vaccine may have certain advantages for the prevention of AOM. Fourth, compared with other available protein vaccines against H. influenzae, the vaccine in this study did not interfere with vaccine components from programmed immunization plans (e.g., TT) and may show similar antigenicity against Hib.

However, in future studies, the coupling conditions need to be further explored and optimized, and the purification process needs to be improved. Bacterial challenge experiments with various serotypes of pneumococcus in control animal experiments need to be performed to verify their cross-immune protection, promote the clinical application of the Hib-PsaA conjugate vaccine, and provide support for immune prevention of AOM and upper respiratory inflammation.

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