Xiaobo Su

Protection against enterovirus 71 with neutralizing epitope incorporation within adenovirus type 3 hexon.

Enterovirus 71 (EV71) is responsible for hand, foot and mouth disease with high mortality among children. Various neutralizing B cell epitopes of EV71 have been identified as potential vaccine candidates. Capsid-incorporation of antigens into adenovirus (Ad) has been developed for a novel vaccine approach. We constructed Ad3-based EV71 vaccine vectors by incorporating a neutralizing epitope SP70 containing 15 amino acids derived from capsid protein VP1 of EV71 within the different surface-exposed domains of the capsid protein hexon of Ad3EGFP, a recombinant adenovirus type 3 (Ad3) expressing enhanced green fluorescence protein. Thermostability and growth kinetic assays suggested that the SP70 epitope incorporation into hypervariable region (HVR1, HVR2, or HVR7) of the hexon did not affect Ad fitness. The SP70 epitopes were thought to be exposed on all hexon-modified intact virion surfaces. Repeated administration of BALB/c mice with the modified Ads resulted in boosting of the anti-SP70 humoral immune response. Importantly, the modified Ads immunization of mother mice conferred protection in vivo to neonatal mice against the lethal EV71 challenge, and the modified Ads-immunized mice serum also conferred passive protection against the lethal challenge in newborn mice. Compared with the recombinant GST-fused SP70 protein immunization, immunization with the Ads containing SP70 in HVR1 or HVR2 elicited higher SP70-specific IgG titers, higher neutralization titers, and conferred more effective protection to neonatal mice. Thus, this study provides valuable information for hexon-modified Ad3 vector development as a promising EV71 vaccine candidate and as an epitope-delivering vehicle for other pathogens.

Construction and characterization of human adenovirus serotype 3 packaged by serotype 7 hexon

Human adenovirus serotype 3 (Ad3) and serotype 7 (Ad7) are important pathogens causing respiratory tract diseases such as acute respiratory disease in pediatric and adult patients, but the immunodominant targets of Ad3- and Ad7-specific neutralizing antibodies (NAbs) remain unclear. A chimeric Ad vector, Ad3/H7, was constructed by replacing the Ad3 hexon gene (H3) with the hexon gene (H7) of Ad7. The chimeric viruses were successfully rescued in HEp-2 cells, and the Ad7 hexon was able to encapsidate the Ad3 genome, and functioned as efficiently as the Ad3 hexon. Furthermore, we tested the host neutralization responses against the viruses using BALB/C mice. Up to 97% of the NAbs produced by mice that were infected with these viruses were specific for the hexon protein in vitro. Preimmunization of mice with one of Ad7 and Ad3/H7 significantly prevented subsequent intranasal infection of the other type in vivo. In contrast, preimmunization of mice with one of Ad3 and Ad3/H7 did not remarkably prevent subsequent infection of the other type. We next evaluated the functional significance of hexon and other structural proteins specific NAbs to suppress the immunogenicity of Ad3/H3 and Ad3/H7 vectors expressing EGFP in mice preimmunized with wild type Ad. Preimmunization of mice with Ad7 evidently suppressed EGFP-specific humoral immune responses elicited by Ad3/H7, and did not exert suppressive effects on Ad3/H3. But contrary to the in vitro neutralization results, EGFP-specific humoral immune responses elicited by Ad3/H7 was remarkably inhibited in Ad3-preimmunization mice. The whole genome of the Ad7 strain was sequenced and aligned with Ad3. The major differences between Ad3 and Ad7 were only observed in the fiber and hexon among all structural proteins, and the variation between the hexons only located in four hypervariable regions (HVRs), HVR-1, -2, -5, and -7. These results thus suggest that Ad3- and Ad7-specific NAbs are directed primarily against the hexon proteins both in vitro and in vivo. But high titer Ad3 fiber-specific NAbs may also play an important role in blunting Ad3 immunogenicity in vivo. These studies contribute to a more profound understanding of Ad immunogenicity and have relevance for the design of novel Ad vaccine.

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

Human adenovirus type 3 (HAdV-3) has produced a global epidemic in recent years causing serious diseases such as pneumonia in both pediatric and adult patients. Development of an effective neutralizing monoclonal antibody (MAb) and identification of its neutralizing epitope is important for the control of HAdV-3 infection. In this study, three neutralizing MAbs were generated, of which MAb 3D7 had a high neutralization titer of 4096 (approximately 0.5 μg/ml) against HAdV-3 infection. In indirect enzyme-linked immunosorbent assays, all three MAbs specifically recognized HAdV-3 virus particles and hexon protein, but did not react with the virus particles or the hexon protein of HAdV-7. Analyses using a series of peptides and chimeric adenovirus particles of epitope mutants revealed that all three MAbs bound to the same exposed region (amino acid positions 244-254 of hexon) in hypervariable region 4 (HVR4), which is highly conserved among global HAdV-3 strains. The amino acids T246 and G250 may be the critical amino acids recognized by these MAbs. MAb 3D7 reduced the recombinant enhanced green fluorescent protein-expressing HAdV-3 (rAd3EGFP) load recovered in the lungs of mice at 3 days post-infection. The generation of MAb 3D7 and the identification of its neutralizing epitope may be useful for therapeutic treatment development, subunit vaccine construction, and virion structural analysis for HAdV-3.

Human Adenovirus Serotype 3 Vector Packaged by a Rare Serotype 14 Hexon.

Recombinant adenovirus serotype 3 (rAd3), which infects cells through the receptor desmoglein 2 (DSG2), has been investigated as a vector for gene therapy or vaccination. However, pre-existing anti-vector immunity may limit the practical application of rAd3. In this study, we investigated the seroprevalence and neutralizing antibody (NAb) titers to Ad3 and alternate serotypes in normal healthy adults in southern China. Sera samples had a high seroprevalence (80.00%) against Ad3 and Ad7 (85.83%), compared with Ad14 (22.50%). Furthermore, 19.17% and 25.83% of samples had high-titer neutralizing antibodies to Ad3 and Ad7, respectively, compared with 3.33% against Ad14. We constructed a chimeric adenovirus, rAd3H14, designed to evade anti-vector immunity by replacing the enhanced green fluorescent protein (EGFP)-expressing hexon of the rAd3EGFP vector with a hexon from Ad14. The chimeric vector rAd3H14 was not neutralized in vitro efficiently by Ad3 NAbs using sera from mice and normal healthy human volunteers. Furthermore, in contrast to the unmodified vector rAd3EGFP, rAd3H14 induced robust antibody responses against EGFP in mice with high levels of pre-existing anti-Ad3 immunity. In conclusion, the chimeric vector rAd3H14 may be a useful alternative vector in adult populations with a high prevalence of Ad3 NAbs.

Prevalence of neutralizing antibodies to common respiratory viruses in intravenous immunoglobulin and in healthy donors in southern China

BACKGROUND Acute respiratory infections (ARIs) are a leading cause of death among children under the age of 5. However, there are no effective drugs for most of these severe viral infections. Passive immunotherapy with convalescent plasma or hyperimmune intravenous immunoglobulin (H-IVIG) is a potential therapeutic option for serious viral infections. It is important to find a suitable source of convalescent plasma and of H-IVIG containing high titer neutralizing antibodies (NAbs). METHODS Sera from 96 healthy adult donors in southern China and commercially available IVIG were analyzed for the titers of NAb to several most common respiratory viruses including respiratory syncytial virus (RSV), seasonal influenza A (InfA), enterovirus 71 (EV71), coxsackievirus A16 (CA16), adenovirus type 3 (Ad3) and a recent epidemic adenovirus type 55 (Ad55) by microneutralization test. RESULTS A high proportion of samples from healthy adult donors were positive for NAbs (>16) to all the viruses except Ad55. A different proportion of these samples had high NAb titers (>512) for InfA (25%), Ad3 (17.71%), RSV (9.38%), EV71 (1.04%), CA16 (3.13%), and Ad55 (4.17%). Commercially available IVIG had high NAb titers to InfA and Ad3 (>1,000) and lower NAb titers to RSV [320], EV71 [160], and CA16 [160]. Strikingly, IVIG also had a high NAb titer to Ad55 (>1,000). CONCLUSIONS Convalescent plasma could be screened from healthy blood volunteers to establish blood banks and to prepare specific H-IVIG for treating severe ARIs caused by common respiratory viruses.

Antigenic variability among two subtypes of human adenovirus serotype 7

Human adenovirus type 7 (HAdV-7) is one of the major serotypes responsible for acute respiratory infection. It is important to investigate the antigenic variabilities of different HAdV-7 genomic subtypes for vaccine development. Phylogenetic analysis of global HAdV-7 strains and major antigen proteins showed that HAdV-7 could be classified into two subtypes. There were three highly variable regions (HVR1, HVR4, and HVR7) in the hexon protein that varied between subtypes. Within each of the subtypes, these regions were conserved. Two subtype HAdV-7 strains isolated in China were used to immunize mice for antigenic characterization. Mice immunized with one subtype strain showed 4–8-fold lower neutralizing antibody titers against another subtype strain. ELISA results showed that the variation in HVR1, 4, and 7 regions contributed to antigenic change, and it may be concluded that the three regions contain subtype-specific epitopes. In summary, strains of HAdV-7 could be divided into two subtypes using genome sequence and antigenic analysis; our results could be important for HAdV-7 vaccine development.