Serotype-Specific Neutralizing Antibody Epitopes of Human Adenovirus Type 3 (HAdV-3) and HAdV-7 Reside in Multiple Hexon Hypervariable Regions

Abstract

bHuman adenovirus types 3 and 7 (HAdV-3 and HAdV-7) occur epidemically and contribute greatly to respiratory diseases, but there is no currently available licensed recombinant HAdV-3/HAdV-7 bivalent vaccine. Identification of serotype-specific neutralizing antibody (NAb) epitopes for HAdV-3 and HAdV-7 will be beneficial for development of recombinant HAdV-3/HAdV-7 bivalent vaccines. In this study, four NAb epitopes within hexon hypervariable regions (HVRs) were predicted for HAdV-3 and HAdV-7, respectively, by using bioinformatics. Eight hexon chimeric adenovirus vectors with the alternation of only one predicted neutralizing epitope were constructed. Further in vitro and in vivo neutralization assays indicated that E2 (residing in HVR2) and E3 (residing in HVR5) are NAb epitopes for HAdV-7, and E3 plays a more important role in generating NAb responses. Cross-neutralization assays indicated that all four predicted epitopes, R1 to R4, are NAb epitopes for HAdV-3, and R1 (residing in HVR1) plays the most important role in generating NAb responses. Humoral immune responses elicited by the recombinant rAdH7R1 (containing the R1 epitope) were significantly and durably suppressed by HAdV-3-specific NAbs. Surprisingly, the rAd⌬E3GFP-specific neutralizing epitope responses induced by rAdMHE3 (R3 replaced by E3) and rAdMHE4 (R4 replaced by E4) were weaker than those of rAdMHE1 (R1 replaced by E1) or rAdMHE2 (R2 relaced by E2) in vitro and in vivo. Furthermore, rAdMHE4 replicated more slowly in HEp-2 cells, and the final yield was about 10-fold lower than that of rAd⌬E3GFP. The current findings contribute not only to the development of new adenovirus vaccine candidates, but also to the construction of new gene delivery vectors.

Construction of New Primer Sets for Corresponding to Genetic Evolution of Human Adenoviruses in Major Capsid Genes through Frequent Recombination

“…The hot spots for recombination are the major capsid genes, namely the hexon, penton base, and fiber genes (17,34). Therefore, the diversity of the major capsid genes, particularly in hexon loops 1 and 2, the penton RGD loop, and the fiber knob, which are associated with binding to neutralizing antibodies and receptors of the host, via recombination may result in escape from the host immune response and the acquisition of different organotropisms in HAdVs (18)(19)(20)(21)(22)(23)(24)(25)(26). Interestingly, our phylogenetic analysis demonstrated that many HAdV types, including our clinical isolates, share the sequences of different types in loop 1 of the hexon, the RGD loop of the penton base, and the fiber knob, thus suggesting evidence of drastic genetic evolution in these regions due to recombination events.…”

“…Additionally, previous reports demonstrated that HAdV-53 and HAdV-64 exhibited close relationships with different proteotypes in these regions (13,16). Since HAdVs contain neutralizing, hemagglutination, and receptor-binding epitopes in loops 1 and 2 of the hexon protein, the RGD loop of the penton protein, and the knob domain of the fiber protein, genomic analyses of these regions provide information on the immunogenicity and tissue tropisms of the HAdVs (18)(19)(20)(21)(22)(23)(24)(25)(26). Furthermore, the variation of these regions through recombination could result in escape from the host immunity and the acquisition of different organotropisms.…”

Identification of a Suppressor Mutation That Improves the Yields of Hexon-Modified Adenovirus Vectors

“…Earlier published studies showed that Ad5 vectors with modifications to only HVR1 (63) or HVR5 (64) avoided Ad5 NAb in vitro and could induce transgene-specific immune responses in animals previously exposed to Ad5. However, more recent work indicates that Ad5-, Ad3-, and Ad7-specific NAb target multiple HVRs (65,66). Ad5-based hexon-modified vectors carrying HVRs 1 to 3, HVR 4, or HVR 5 of Ad48 underperformed relative to an Ad5 vector containing all HVRs of Ad48, indicating that modification to multiple HVRs is necessary for full immune avoidance in vivo (65).…”

Mapping the epitope of neutralizing monoclonal antibodies against human adenovirus type 3

“…The following strains of adenoviruses used in this study were obtained as previously described (Tian et al, 2011;Qiu et al, 2012;Zhang et al, 2009): recombinant adenovirus rAd3EGFP encoding a HAdV-3 GZ-01 genome and an enhanced green fluorescent protein (eGFP) with an E3 region deletion, hexon chimeric adenovirus rAd3egf/H7 generated by replacing the HAdV-3 hexon gene of the rAd3EGFP with the hexon gene from HAdV-7 GZ08 strain, epitope mutants (rAd3H7R1, rAd3H7R2, rAd3H7R5, and rAd3H7R7) from rAd3egf/H7 replaced with corresponding HAdV-3 epitopes, and epitope chimeric mutant rAdMHE3 from rAd3EGFP replaced with HVR5 of HAdV-7. All the adenoviruses were cultured in HEp-2 cells or AD293 cells as previously described (Tian et al, 2011).…”

“…additional highly variable regions (Rux et al, 2003;Bradley et al, 2012;Yuan et al, 2009). Our previous study demonstrated that HVR1, 2, 5, and 7 of HAdV-3 contained neutralizing epitopes (Qiu et al, 2012).…”