Recombinant hemagglutinin produced from Chinese Hamster Ovary (CHO) stable cell clones and a PELC/CpG combination adjuvant for H7N9 subunit vaccine development

Abstract

The novel H7N9 avian influenza A virus has caused human infections in China since 2013; some isolates from the fifth wave of infections have emerged as highly pathogenic avian influenza viruses. Recombinant hemagglutinin proteins of H7N9 viruses can be rapidly and efficiently produced with low-level biocontainment facilities. In this study, recombinant H7 antigen was obtained from engineered stable clones of Chinese Hamster Ovary (CHO) cells for subsequent large-scale production. The stable CHO cell clones were also adapted to grow in serum-free suspension cultures. To improve the immunogenicity of the recombinant H7 antigens, we evaluated the use of a novel combination adjuvant of PELC and CpG (PELC/CpG) to augment the anti-H7N9 immune responses in mice. We compared the effects with other adjuvants such as alum, AddaVax (MF59-like), and several Toll-like receptor ligands such as R848, CpG, and poly (I:C). With the PELC/CpG combination adjuvant, CHO cell-expressed rH7 antigens containing terminally sialylated complex type N-glycans were able to induce high titers of neutralizing antibodies in sera and conferred protection following live virus challenges. These data indicate that the CHO cell-expressed recombinant H7 antigens and a PELC/CpG combination adjuvant can be used for H7N9 subunit vaccine development.

Keywords: CHO cells; H7N9 vaccine; Hemagglutinin; PELC/CpG adjuvant.

Fig. 1

Expression and characterization of rH7 in stable clones of CHO cells. (A) CHO cell vector for rH7 expression; (B) purified rH7 protein analysis on SDS-PAGE gels with Coomassie blue staining; (C) Western blot analysis of purified rH7 protein after Endo H and PNGase F treatments.

Fig. 2

Total IgG titers elicited by intramuscular immunization with rH7 formulated with PELC/CpG combination adjuvant. (A) Groups of BALB/c mice were intramuscularly immunized with CHO-rH7 formulated with alum, R848, CpG, AddaVax, poly (I:C), or PELC/CpG adjuvant for two doses within a 3-week interval. Total IgG titers in sera were determined by ELISA for (B) 0.2 µg and (C) 2 µg CHO-rH7 antigen without or with alum, R848, CpG, AddaVax, poly (I:C), or PELC/CpG adjuvant. * indicates P < 0.05; ** indicates P < 0.01, ***, indicates P < 0.001.

Fig. 3

IgG1, IgG2a, and IgG2b subtypes in sera. IgG1 titers in antisera from mice immunized with the formulation of alum, R848, CpG, AddaVax, poly (I:C), or PELC/CpG adjuvant with (A) 0.2 µg and (D) 2 µg dose of CHO-rH7 antigen. The IgG2a titers in antisera from mice immunized with the formulation of alum, R848, CpG, AddaVax, poly (I:C), or PELC/CpG adjuvant with (B) 0.2 µg and (E) 2 µg dose of CHO-rH7 antigen. The IgG2b titers in antisera from mice immunized with the formulation of alum, R848, CpG, AddaVax, poly (I:C), or PELC/CpG adjuvant with (C) 0.2 µg and (F) 2 µg dose of CHO-rH7 antigen. * indicates P < 0.05; ** indicates P < 0.01, ***, indicates P < 0.001.

Fig. 4

HI and MN antibody titers in sera. HI titers in antisera from mice immunized with the formulation of alum, R848, CpG, AddaVax, poly (I:C), or PELC/CpG adjuvant with (A) 0.2 µg and (B) 2 µg dose of CHO-rH7 antigen. The MN titers in antisera from mice immunized with the formulation of alum, R848, CpG, AddaVax, poly (I:C), or PELC/CpG adjuvant with (C) 0.2 µg and (D) 2 µg dose of CHO-rH7 antigen. * indicates P < 0.05; ** indicates P < 0.01, ***, indicates P < 0.001.

Fig. 5

Protective immunity against the H7N9 virus. Three weeks after second dose immunization, mice were anesthetized and subjected to intranasal challenge with 10 LD50 of H7N9 virus (A/Taiwan/01/2013) in a volume of 50 μL. (A) Survival rate and (B) body weight loss were monitored for 14 days. Mice whose body weights fell below 75% of their initial weights were sacrificed.

Fig. 6

Immunogenicity of CHO-rH7 with different N-glycan patterns. (A) purified CHO-rH7, CHO-rH7 (KIF), and CHO-rH7 (KIF + E) protein analysis on SDS-PAGE gels with Coomassie blue staining; (B) MN titers (ID-50) in antisera from mice immunized with 0.2 µg CHO-rH7, CHO-rH7 (KIF), or CHO-rH7 (KIF + E) antigen plus PELC/CpG adjuvant; (C) Survival rate and (D) body weight loss from immunized mice following live virus challenge were monitored for 14 days. Mice whose body weights fell below 75% of their initial weights were sacrificed.

Fig. 7

Stable CHO cell clone in serum-free suspension cultures. Cell density and rH7 production titer of the adapted stable CHO cell clones cultured in serum-free suspension cultures. The culture medium was completely replaced with fresh medium at day 3 and 6 to maintain the cell viability around 90%.