Haemophilus influenzae type b (Hib) is a widely recognized member of the
Haemophilus genus that directly causes respiratory infectious disease with characteristic manifestations of tympanitis, bronchitis and pneumonia in children of all ages, particularly in infants and 1-year-olds [
1‐
3]. Notably, this pathogen is associated with purulent meningitis in a specific ratio in all Hib-infected patients [
1,
4]. Epidemiological studies of this pathogen have primarily been performed in developed countries and have shown that the incidence in children under the age of 5 in areas such as the US, France, and Switzerland is approximately 20–100/100,000 [
5‐
9], but few data have been reported for developing areas. Although no systematic, epidemiological study of Hib has been performed in mainland China, studies of this pathogen in a small number of populations in different regions of China have indicated that the potential incidence of pediatric infections in mainland China is noteworthy [
10,
11]. Numerous studies have addressed Hib structure and immunology [
12,
13], leading to the licensure of a preventive vaccine against Hib infection in the 1980s [
1]. Based on the results of a structural study of the Hib agent that suggested that the bacterium presented a capsular polysaccharide (CPS) antigen and thallus antigen, the early vaccine was prepared with polyribosylribitol phosphate (PRP), which is composed of ribosylribitol phosphate as the basic unit [
14,
15]. The CPS antigen is the primary component of the 1st generation Hib vaccine and has been widely used to vaccinate children. Clinical observation of the use of this vaccine in pediatric populations indicated that it induced a remarkable immune response in children 18 months of age or older but did not provoke a satisfactory response in children younger than 18 months [
16‐
19]. Subsequent Hib conjugate vaccines were developed based on a CPS antigen-binding protein (i.e., diphtheria toxoid (DT), tetanus toxoid (TT) and the
N. meningitidies outer membrane protein (OMP) Hib thallus antigen protein) [
18,
20]. Previous studies of this conjugate vaccine in mice and macaques have shown that remarkable immunity is induced by immunization with this vaccine compared to that induced by the CPS vaccine; the immunity usually presents as an increased antibody response in serum [
21]. The results of additional clinical trials comparing conjugate Hib vaccines produced with various carrier proteins, including DT, TT and OMP, suggested that one or two immunizations induced a lower antibody response in children of various ages than three immunizations [
22,
23]. The data indicated that the antibody levels of Hib vaccines conjugated with DT, TT, and OMP were 0.06, 0.05, and 0.83 μg/ml, respectively, after the first inoculation and that the levels increased to 0.14, 0.26, and 1.22 μg/ml after the second inoculation [
23]. However, the levels increased further to 0.28, 3.64, and 1.14 μg/ml after the third inoculation [
23]. Based on these data, a routine immunization schedule for the Hib conjugate vaccine has been recommended for children worldwide by the WHO [
24], and the vaccines have been used extensively in multiple countries, including China [
25,
26]. The increased immunogenicity associated with the Hib conjugate vaccine compared with that of the CPS vaccine suggests that binding of the semi-antigen CPS to the carrier protein would provide an effective antigen for immunization of individuals [
27‐
30].
In previous studies, the immunogenicity of Hib CPS antigen and its protein conjugates were studied in juvenile and infant rhesus macaques [
31,
32], which suggest that the conjugate of CPS and TT, DT and OMP are capable of inducing stronger specific antibody responses than CPS alone in this animal model [
31‐
33]. Although those studies indicated unequal level of antibody induced against various conjugate vaccine including Hib-TT, Hib-CT and Hib OMPC and confirmed their immunogenicity, but also suggested the role of these carrier protein in the immunogenicity of Hib conjugate vaccine in human should be studied further [
33,
34]. To better understand the immunologic mechanism of this conjugate vaccine, especially the role of carrier protein to enhance immunogenicity of CPS via the immunization schedule of three doses, we conducted a study using rhesus macaques, which are closely genetically related to humans, in which we assessed the mRNA profiles of rhesus macaque peripheral blood mononuclear cells (PBMCs) after immunization of the animals with the Hib conjugate vaccine, the Hib polysaccharide antigen or the carrier protein TT. Using comparison and analysis of these mRNA gene profiles, we investigated the molecular mechanism of immunity induced by the Hib conjugate vaccine and identified several genes that are likely to play a role in specific and effective immune responses against Hib in rhesus macaques. These genes might be induced by CPS antigen and carrier protein and might play the roles involved in the enhancement of immunogenicity of Hib conjugate vaccine.