Wild-Type Human Adenovirus (Serotype 4)

Human adenovirus type 4 (HAdV-4) is an epidemic virus that causes severe acute respiratory disease (ARD) in both pediatric and adult patients. However, no licensed drug or vaccine is currently available to the civilian population. The identification of neutralizing epitopes for HAdV-4 will aid in the development of novel antiviral vaccines and gene transfer vectors, and potent neutralizing monoclonal antibodies (MAbs) will aid in the development of suitable antiviral drugs. This study reports that MAb MN4b exhibits potent neutralizing activity against HAdV-4. MN4b recognizes a conformational epitope (418AGSEK422) within hypervariable region 7 (HVR7). Mutations at this site enable HAdV-4 mutants to escape neutralization by MN4b and resist neutralization by animal and human anti-HAdV-4 sera. The recombinant virus rAd3-A4R7-1, which contains the identified neutralizing epitope in the HVR7 region of the HAdV-3 hexon, successfully induced antiserum that inhibited HAdV-4 infection. These results suggest that a small surface loop of the HAdV-4 hexon is a key neutralizing epitope for the virus. The generation of MN4b and the identification of this neutralizing epitope may aid in the development of therapeutics, subunit vaccines, and novel vectors that can evade preexisting neutralization of HAdV-4.

HAdV-4 HVR7 can be divided into two independent regions: R7-1 and R7-2. To map the neutralizing epitopes recognized by MN4b in an independent manner, three recombinant type 4 adenoviruses were constructed and mutations were introduced in the long loop of HVR7 (Figure 1A). The replication titers and particle/PFU ratios of these mutant viruses were similar to those of wild-type human adenovirus type 4 (HAdV-4), indicating that the mutation in R7 did not significantly change the structure of the virion. Subsequently, these mutant viruses were screened for MN4b using ELISA, Western blotting, and neutralization assays. Mutations in the R7-2 region (mutant rAd4-3R2) did not affect the recognition and neutralization of the recombinant virus by MN4b (Figure 1). However, mutants rAd4-3R7-1 and rAd4-3R7 (with altered residues in R7-1, AGSEK to DANG) escaped neutralization by MN4b (Figure 1D). ELISA and Western blot data also showed that rAd4-3R7-1 and rAd4-3R7 completely escaped recognition by MN4b (Figure 1B and C). MAb 3D7, a neutralizing antibody against HAdV-3 prepared previously, was used as the control. These results confirmed that the R7-1 residue (AGSEK) constitutes a key epitope recognized by MN4b.

Figure 1. Confirmation of the epitope recognized by MN4b with mutations in HVR7 of HAdV-4. (Tian X, et al., 2018)