Prime-boost immunization with inactivated human adenovirus type 55 combined with an adjuvant enhances neutralizing antibody responses in mice

Abstract

Background: Human adenovirus type 55 (hAd55) infection can lead to acute respiratory diseases that often present with severe symptoms. Despite its persistent prevalence in military camps and communities, there are no commercially available vaccines or vaccine candidates undergoing clinical evaluation; therefore, there is an urgent need to address this. In this study, we evaluated the immunogenicity of inactivated hAd55 isolates and investigated the effects of adjuvants and various immunization intervals.

Methods and results: To select a vaccine candidate, four hAd55 strains (6-9, 6-15 (AFMRI 41014), 28-48 (AFMRI 41013), and 12-164 (AFMRI 41012)) were isolated from infected patients in military camps. Sequence analysis revealed no variation in the coding regions of structural proteins, including pentons, hexons, and fibers. Immunization with inactivated hAd55 isolates elicited robust hAd55-specific binding and neutralizing antibody responses in mice, with adjuvants, particularly alum hydroxide (AH), enhancing antibody titers. Co-immunization with AH also induced hAd14-specific neutralizing antibody responses but did not induce hAd11-specific neutralizing antibody responses. Notably, booster immunization administered at a four-week interval resulted in superior immune responses compared with shorter immunization intervals.

Conclusions: Prime-boost immunization with the inactivated hAd55 isolate and an AH adjuvant shows promise as a potential approach for preventing hAd55-induced respiratory disease. Further research is needed to evaluate the efficacy and safety of these vaccine candidates in preventing hAd55-associated respiratory illnesses.

Keywords: Acute respiratory disease; Human adenovirus type 55; Inactivated viral vaccine; Neutralizing antibody; Respiratory infection.

Fig. 1

Isolation and genotyping of human adenovirus type 55 (hAd55). (A) Conventional PCR for vaccine candidates. (B) Real-time PCR using SYBR. (red: negative control (NC); yellow: 6–9; dark blue: 6–15 (AFMRI 41014); sea blue: 12–164 (AFMRI 41012); blue: 28–48 (AFMRI 41013)). (C) The cytopathic effects (CPE) in A549 cells induced by hAd55. (D) Real-time PCR using a TaqMan probe for the hAd55-specific primer. (red: negative control (NC); yellow: 6–9; dark blue: 6–15 (AFMRI 41014); sea blue: 12–164 (AFMRI 41012); blue: 28–48 (AFMRI 41013)). Neg.: negative control

Fig. 2

Phylogenetic analysis of the hAd55 isolates. (A) Complete genomes of adenovirus genotypes. (B) Phylogenetic tree for hAd55 isolates. (C) Single nucleotide polymorphism (SNP) analysis of hAd55 isolates. (D) The specific locations of SNPs within the genomic nucleotide sequence of hAd55

Fig. 3

Immunogenicity assessment of inactivated hAd55 (6–15 (AFMRI 41014)) at three-different doses. Mice were immunized intramuscularly with 5 × 10⁵, 1 × 10⁶, or 2 × 10⁶ pfu of inactivated hAd55 (6–15 (AFMRI 41014) two times via two-week interval (A). hAd55-specific binding antibody titers (B) and neutralizing antibody titers (C) were analyzed by ELISA or PRNT using sera collected two weeks after the first and second immunizations (for ELISA) or after the second immunization (for PRNT)

Fig. 4

Immunogenicity assessment of inactivated hAd55 isolates. Mice were immunized intramuscularly with PBS or hAd55 isolates (6–9, 6–15 (AFMRI 41014), 28–48 (AFMRI 41013), or 12–164 (AFMRI 41012)), with or without Imject alum, twice at two-week intervals. (A) To measure hAd55-specific binding antibody titers, sera were collected two weeks after each immunization, and an enzyme-linked immunosorbent assay (ELISA) was performed. (B) To assess hAd55-specific neutralizing antibody (nAb) titers, the plaque reduction neutralization test (PRNT) was performed using sera collected two weeks after the second immunization. N.D.: not detected, *P < 0.05, **P < 0.01

Fig. 5

Comparison of adjuvants for inactivated hAd55. Mice were immunized intramuscularly with the hAd55 isolate 6–15 (AFMRI 41014), with or without adjuvants (Imject, AddaVax, dmLT, AH, or AP), or PBS twice at a two-week interval. Various amounts of alum adjuvant were tested for AH and AP, as described in the Materials and Methods section. The adjuvant effects were assessed in terms of hAd55-specific binding antibody titers (A) and nAb titers (B). Neutralizing antibody titers were assessed using sera collected 2 weeks after the second immunization. AH: alum hydroxide; AP: alum phosphate; N.D.: not detected. *P < 0.05 vs. 6–15 (AFMRI 41014), **P < 0.01 vs. 6–15 (AFMRI 41014)

Fig. 6

Assessment of hAd11 and hAd14-specific nAb induction following immunization with inactivated hAd55 combined with adjuvants. Sera from mice immunized with 6–15 (AFMRI 41014) plus AH (1:1/7) were assessed for hAd11- and hAd14-specific nAb titers using a PRNT. Bar graph (A) and table (B) for PRNT₅₀ values of hAd11, hAd14, and hAd55. N.D.: not detected

Fig. 7

Comparison of immunization intervals for inactivated hAd55. (A) Mice were immunized intramuscularly with the hAd55 isolate (6–15 (AFMRI 41014)), with or without AH, twice at one-, two-, or four-week intervals, and sera were collected at the indicated time points. (B) hAd55-specific binding antibodies were assessed by ELISA in sera collected 0, 2, and 4 weeks after the last immunization. (C) hAd55-specific nAb titers were assessed by PRNT in sera collected two weeks after the last immunization. AH: alum hydroxide, N.D.: not detected, *P < 0.05, **P < 0.01