Neutralization Antigenic Site Research Articles

Identification of four neutralizing antigenic sites on the enterovirus D68 capsid.

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 βB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.

Neutralization of African enterovirus A71 genogroups by antibodies to canonical genogroups.

Enterovirus 71 (EV-A71) is a major public health problem, causing a range of illnesses from hand-foot-and-mouth disease to severe neurological manifestations. EV-A71 strains have been phylogenetically classified into eight genogroups (A to H), based on their capsid-coding genomic region. Genogroups B and C have caused large outbreaks worldwide and represent the two canonical circulating EV-A71 subtypes. Little is known about the antigenic diversity of new genogroups as compared to the canonical ones. Here, we compared the antigenic features of EV-A71 strains that belong to the canonical B and C genogroups and to genogroups E and F, which circulate in Africa. Analysis of the peptide sequences of EV-A71 strains belonging to different genogroups revealed a high level of conservation of the capsid residues involved in known linear and conformational neutralization antigenic sites. Using a published crystal structure of the EV-A71 capsid as a model, we found that most of the residues that are seemingly specific to some genogroups were mapped outside known antigenic sites or external loops. These observations suggest a cross-neutralization activity of anti-genogroup B or C antibodies against strains of genogroups E and F. Neutralization assays were performed with diverse rabbit and mouse anti-EV-A71 sera, anti-EV-A71 human standards and a monoclonal neutralizing antibody. All the batches of antibodies that were tested successfully neutralized all available isolates, indicating an overall broad cross-neutralization between the canonical genogroups B and C and genogroups E and F. A panel constituted of more than 80 individual human serum samples from Cambodia with neutralizing antibodies against EV-A71 subgenogroup C4 showed quite similar cross-neutralization activities between isolates of genogroups C4, E and F. Our results thus indicate that the genetic drift underlying the separation of EV-A71 strains into genogroups A, B, C, E and F does not correlate with the emergence of antigenically distinct variants.

Epidemiological and Genetic Analysis of Foot-and-Mouth Disease Virus O/ME-SA/Ind-2001 in China between 2017 and 2021

The O/ME-SA/Ind-2001 lineage of foot-and-mouth disease virus (FMDV) was introduced into China in 2017, which subsequently caused 19 outbreaks of foot-and-mouth disease (FMD) and emerged in 8 provinces in China between 2017 and 2021. It is the first time for WOAH/national reference laboratory for FMD (LVRI) to comprehensively summarize these 19 outbreaks of O/ME-SA/Ind-2001 for consecutive 5 years. Our study selected and conducted whole viral genome sequencing for 9 representative isolates and the VP1 sequences of the FMDV-positive samples collected between 2017 and 2021. Analyses of these gene sequences showed that all the field strains belonged to O/ME-SA/Ind-2001e. Phylogenetic analysis indicated that these viruses were closely related to those circulating in neighboring countries, and there were at least 3 different FMD viral clades (named cluster 1, cluster 2, and cluster 3) circulating during this period. Also, a gradually decreasing nucleotide identity was observed in newly emerging viruses year by year compared with the first isolate identified in 2017. These results suggest extensive movements and constant and rapid evolvement of the O/ME-SA/Ind-2001e sublineage. Besides, the neutralizing antigenic sites in the structural proteins for these O/ME-SA/Ind-2001e viruses were analyzed to predict the vaccine matching between these strains and the commercial vaccine strain O/BY/CHA/2010. The results showed that the VP1 epitope 135–144, highly associated with neutralizing antibody induction, was variable among these strains. The mutations in this region reflected a potential risk of challenging the current vaccine protection. Therefore, there is an urgent need to reinforce the importance of improved FMD surveillance and monitor the evolution of O/ME-SA/Ind-2001e, which will provide essential information for the FMD control program in China and Asia.

Receptor binding motif surrounding sites in the spike 1 protein of infectious bronchitis virus have high susceptibility to mutation related to selective pressure.

To date, various genotypes of infectious bronchitis virus (IBV) have co-circulated and in Korea, GI-15 and GI-19 lineages were prevailing. The spike protein, particularly S1 subunit, is responsible for receptor binding, contains hypervariable regions and is also responsible for the emerging of novel variants. This study aims to investigate the putative major amino acid substitutions for the variants in GI-19. The S1 sequence data of IBV isolated from 1986 to 2021 in Korea (n = 188) were analyzed. Sequence alignments were carried out using Multiple alignment using Fast Fourier Transform of Geneious prime. The phylogenetic tree was generated using MEGA-11 (ver. 11.0.10) and Bayesian analysis was performed by BEAST v1.10.4. Selective pressure was analyzed via online server Datamonkey. Highlights and visualization of putative critical amino acid were conducted by using PyMol software (version 2.3). Most (93.5%) belonged to the GI-19 lineage in Korea, and the GI-19 lineage was further divided into seven subgroups: KM91-like (Clade A and B), K40/09-like, QX-like (I-IV). Positive selection was identified at nine and six residues in S1 for KM91-like and QX-like IBVs, respectively. In addition, several positive selection sites of S1-NTD were indicated to have mutations at common locations even when new clades were generated. They were all located on the lateral surface of the quaternary structure of the S1 subunits in close proximity to the receptor-binding motif (RBM), putative RBM motif and neutralizing antigenic sites in S1. Our results suggest RBM surrounding sites in the S1 subunit of IBV are highly susceptible to mutation by selective pressure during evolution.

Genetic characterization and distribution of the virus in chicken embryo tissues infected with Newcastle disease virus isolated from commercial and native chickens in Indonesia.

Background and Aim:Newcastle disease (ND) is a viral infectious disease that affects commercial and native chickens, resulting in economic losses to the poultry industry. This study aimed to examine the viral strains circulating in commercial and native chickens by genetic characterization and observe the distribution of Newcastle disease virus (NDV) in chicken embryonic tissue.Materials and Methods:ND was detected using a quantitative reverse transcription-polymerase chain reaction. Genetic characterization of the fusion (F) and hemagglutinin-neuraminidase (HN) genes from the eight NDVs was performed using specific primers. The sequence was compared with that of other NDVs from GenBank and analyzed using the MEGA-X software. The distribution of NDV in chicken embryos was analyzed based on lesions and the immunopositivity in immunohistochemistry staining.Results:Based on F gene characterization, velogenic NDV strains circulating in commercial and native chickens that showed varying clinical symptoms belonged to genotype VII.2. Lentogenic strains found in chickens without clinical symptoms were grouped into genotype II (unvaccinated native chickens) and genotype I (vaccinated commercial chickens). Amino acid variations in the HN gene, namely, the neutralization epitope and antigenic sites at positions 263 and 494, respectively, occurred in lentogenic strains. The NDV reaches the digestive and respiratory organs, but in lentogenic NDV does not cause significant damage, and hence embryo death does not occur.Conclusion:This study showed that velogenic and lentogenic NDV strains circulated in both commercial and native chickens with varying genotypes. The virus was distributed in almost all organs, especially digestive and respiratory. Organ damage in lentogenic infection is not as severe as in velogenic NDV. Further research is needed to observe the distribution of NDV with varying pathogenicity in chickens.

Molecular Basis of Antigenic Drift in Serotype O Foot-and-Mouth Disease Viruses (2013-2018) from Southeast Asia.

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals with serious economic consequences. FMD is endemic in Southeast Asia (SEA) and East Asia (EA) with the circulation of multiple serotypes, posing a threat to Australia and other FMD-free countries. Although vaccination is one of the most important control measures to prevent FMD outbreaks, the available vaccines may not be able to provide enough cross-protection against the FMD viruses (FMDVs) circulating in these countries due to the incursion of new lineages and sub-lineages as experienced in South Korea during 2010, a FMD-free country, when a new lineage of serotype O FMDV (Mya-98) spread to the country, resulting in devastating economic consequences. In this study, a total of 62 serotype O (2013–2018) viruses selected from SEA and EA countries were antigenically characterized by virus neutralization tests using three existing (O/HKN/6/83, O/IND/R2/75 and O/PanAsia-2) and one putative (O/MYA/2009) vaccine strains and full capsid sequencing. The Capsid sequence analysis revealed three topotypes, Cathay, SEA and Middle East-South Asia (ME-SA) of FMDVs circulating in the region. The vaccines used in this study showed a good match with the SEA and ME-SA viruses. However, none of the recently circulating Cathay topotype viruses were protected by any of the vaccine strains, including the existing Cathay topotype vaccine (O/HKN/6/83), indicating an antigenic drift and, also the urgency to monitor this topotype in the region and develop a new vaccine strain if necessary, although currently the presence of this topotype is mainly restricted to China, Hong Kong, Taiwan and Vietnam. Further, the capsid sequences of these viruses were analyzed that identified several capsid amino acid substitutions involving neutralizing antigenic sites 1, 2 and 5, which either individually or together could underpin the observed antigenic drift.

Type I vaccine-derived polioviruses in China from 1995 to 2019

A nation-wide case surveillance was conducted in China since 1995 for the objective of identifying acute flaccid paralysis (AFP) in children so that potential wild polioviruses and vaccine-derived poliovirus (VDPV) could be identified on time. Two outbreaks associated with type I circulating VDPVs, eight native independent type I ambiguous VDPVs (aVDPV), and one imported aVDPV were identified during the AFP case surveillance in China from 1995 to 2019. The VP1 coding region of the Chinese type I VDPVs differed from the polio vaccine strain by 1.00%–3.75% (9–34 substitutions in 906 nucleotides). Most of the Chinese type I VDPV strains shared 4 amino acid substitutions in the neutralizing antigenic (NAg) sites: 3 located at the BC loop, which formed the NAg site 1, and another at NAg site 3a. All of the Chinese type I VDPVs identified during the AFP case surveillance were young VDPVs, which indicated a limited viral replication resulted from the administration of the initiating oral polio vaccine (OPV) dose. VDPVs can emerge and spread in isolated communities with immunity gaps and the circulation ceases following a mass immunization with OPV. As such, high-quality surveillance permitted very early detection and response and it played a key role in stalling the widespread circulation of the emergent cVDPV strains in China.

The Optimal Concentration of Formaldehyde is Key to Stabilizing the Pre-Fusion Conformation of Respiratory Syncytial Virus Fusion Protein.

Background: To date, there is no licensed vaccine available to prevent respiratory syncytial virus (RSV) infection. The valuable pre-fusion conformation of the fusion protein (pre-F) is prone to lose high neutralizing antigenic sites. The goals of this study were to stabilize pre-F protein by fixatives and try to find the possibility of developing an inactivated RSV vaccine. Methods: The screen of the optimal fixative condition was performed with flow cytometry. BALB/c mice were immunized intramuscularly with different immunogens. The serum neutralizing antibody titers of immunized mice were determined by neutralization assay. The protection and safety of these immunogens were assessed. Results: Fixation in an optimal concentration of formaldehyde (0.0244%–0.0977%) or paraformaldehyde (0.0625%–1%) was able to stabilize pre-F. Additionally, BALB/c mice inoculated with optimally stabilized pre-F protein (opti-fixed) induced a higher anti-RSV neutralization (9.7 log2, mean value of dilution rate) than those inoculated with unstable (unfixed, 8.91 log2, p < 0.01) or excessively fixed (exce-fixed, 7.28 log2, p < 0.01) pre-F protein. Furthermore, the opti-fixed immunogen did not induce enhanced RSV disease. Conclusions: Only the proper concentration of fixatives could stabilize pre-F and the optimal formaldehyde condition provides a potential reference for development of an inactivated RSV vaccine.

Identification of novel epitopes in serotype O foot-and-mouth disease virus by in vitro immune selection.

Foot-and-mouth disease virus (FMDV) displays various epitopes on the capsid outer surface. In addition to the five neutralizing antigenic sites, there is evidence of the existence of other, yet unidentified, epitopes that are believed to play a role in antibody-mediated protection. Previous attempts to identify these epitopes revealed two additional substitutions at positions VP2-74 and -191 (5M2/5 virus) to be of antigenic significance. However, complete resistance to neutralization was not obtained in the neutralization assay, indicating the existence of other, undisclosed epitopes. Results from this study provides evidence of at least two new neutralizing epitopes involving residues VP3-116 and -195 around the threefold axis that have significant impact on the antigenic nature of the virus. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and should help with rational vaccine design.

Recombinant virus-like particle presenting a newly identified coxsackievirus A10 neutralization epitope induces protective immunity in mice

Coxsackievirus A10 (CVA10) has emerged as one of the major pathogens of hand, foot, and mouth disease in recent years. However, there are no approved vaccines or effective drugs against CVA10. Several experimental CVA10 vaccines have been shown to elicit neutralizing antibodies that could confer protection against viral infection. However, neutralizing antigenic sites on CVA10 capsid have not been well characterized. Here, we report the characterization of linear neutralization epitopes of CVA10 and the development of a CVA10 vaccine based on the identified epitopes. We showed that peptide VP2-P28, corresponding to residues 136 to 150 of VP2, were recognized by anti-inactivated CVA10 sera and effectively inhibited anti-CVA10 sera-mediated neutralization, suggesting that this peptide contains neutralizing epitopes. Insertion of VP2-P28 into hepatitis B core antigen (HBc) resulted in a chimeric virus-like particle (VLP; designated HBc-P28) with the CVA10 epitope exposed on the particle surface. HBc-P28 VLP elicited strong antibody responses against VP2-P28 in mice. Anti-HBc-P28 sera could neutralize both CVA10 clinical isolates and prototype strain, consistent with the fact that the VP2-P28 sequence is highly conserved among CVA10 strains. In addition, anti-HBc-P28 sera failed to cross-neutralize other HFMD-causing enteroviruses, indicating that neutralizing antibodies elicited by HBc-P28 VLP were CVA10-specific. Importantly, anti-HBc-P28 sera were able to provide efficient protection against lethal CVA10 infection in recipient mice. Collectively, these data show that peptide VP2-P28 represents a CVA10-specific linear neutralizing antigenic site and chimeric VLP displaying this peptide is a promising epitope-based CVA10 vaccine candidate.

Genetic and antigenic relationship of foot–and–mouth disease virus serotype O isolates with the vaccine strain O1/BFS

Foot–and–mouth disease serotype O viruses (FMDV/O) are responsible for the most outbreaks in FMD endemic countries. O1/BFS is one of the recommended FMD/O vaccine strains by World Reference Laboratory for FMD. In the current study, FMDV/O1 BFS vaccine strain and serotype O field isolates (45) were analyzed phylogenetically and antigenically to gain more insight into the genetic and antigenic characteristics of the vaccine strain and field isolates.O1/BFS showed similarity with 89% of the field isolates using a virus neutralization test (VNT). The P1 region encoding the FMDV capsid was sequenced and analysed for 46 strains of FMDV/O. Phylogenetic analysis showed these viruses originated from five continents and covered eight of 11 reported topotypes. Five isolates that demonstrated low antigenic similarities with O1/BFS were analyzed for their antigenic variation at the known neutralizing antigenic sites. Three of the five isolates demonstrated unique amino acid substitutions at various antigenic sites. No unique amino acid substitutions were observed for the other two unmatched isolates. Positively selected residues were identified on the surface of the FMD virus capsid supporting that it is important to continuously monitor field isolates for their antigenic and phenotypic changes.In conclusion, the vaccine strain O1/BFS is likely to confer protection against 89% of the 45 FMDV/O isolates based on VNT. Thus O1/BFS vaccine strain is still suitable for use in global FMD serotype O outbreak control. Combining data from phylogenetic, molecular and antigenic analysis can provide improvements in the process of vaccine selection.

Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2-MPER region.

Summary paragraphEbola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMappTM that has been shown to be effective in nonhuman primate models of infection1 and has been used under compassionate-treatment protocols in humans2. ZMappTM is a mixture of three chimerized murine monoclonal antibodies (mAbs)3–6 that target EBOV-specific epitopes on the surface glycoprotein (GP)7,8. However, ZMappTM mAbs do not neutralize other species from the Ebolavirus genus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here we describe three naturally-occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane proximal external region (MPER) of GP. The identification of a conserved neutralizing antigenic site in the GP suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2/MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

Designing a Novel Recombinant HN Protein with Multi Neutralizing Antigenic Sites and Auto Tag Removal Ability Based on NDV-VIIj for Diagnosis and Vaccination Application

Hemagglutinin-neuraminidase (HN) protein besides its mediation in viral pathogenesis, is composed of various antigenic sites which stimulate production of host's antibodies. Thus, application of this protein in serological tests and vaccination plays a major role in biosecurity and control programs. In the present study, we designed a recombinant HN protein containing different neutralizing antigenic sites with velogenic patterns, and sub-cloned it into pET-43.1a+ expression vector. The expression of NusA-HN recombinant protein was induced. Affinity chromatography protein purification using HisPur™ Ni-NTA was then conducted. Moreover, we performed western-blot technique using HRP-conjugated Anti His-Tag. Results revealed that following induction of recombinant protein, two distinct bands of HN-61kDa and NusA-63kDa were purified and identified by western-blotting. We recommend further analysis should be carried out to determine the functional role of this recombinant protein in enzyme-linked immunosorbent assays for Newcastle disease diagnosis. This HN protein containing multi neutralizing antigenic sites might also be applicable in vaccination programs to increase vaccines potency.

Dynamics of Evolution of Poliovirus Neutralizing Antigenic Sites and Other Capsid Functional Domains during a Large and Prolonged Outbreak.

We followed the dynamics of capsid amino acid replacement among 403 Nigerian outbreak isolates of type 2 circulating vaccine-derived poliovirus (cVDPV2) from 2005 through 2011. Four different functional domains were analyzed: (i) neutralizing antigenic (NAg) sites, (ii) residues binding the poliovirus receptor (PVR), (iii) VP1 residues 1 to 32, and (iv) the capsid structural core. Amino acid replacements mapped to 37 of 43 positions across all 4 NAg sites; the most variable and polymorphic residues were in NAg sites 2 and 3b. The most divergent of the 120 NAg variants had no more than 5 replacements in all NAg sites and were still neutralized at titers similar to those of Sabin 2. PVR-binding residues were less variable (25 different variants; 0 to 2 replacements per isolate; 30/44 invariant positions), with the most variable residues also forming parts of NAg sites 2 and 3a. Residues 1 to 32 of VP1 were highly variable (133 different variants; 0 to 6 replacements per isolate; 5/32 invariant positions), with residues 1 to 18 predicted to form a well-conserved amphipathic helix. Replacement events were dated by mapping them onto the branches of time-scaled phylogenies. Rates of amino acid replacement varied widely across positions and followed no simple substitution model. Replacements in the structural core were the most conservative and were fixed at an overall rate ∼20-fold lower than the rates for the NAg sites and VP1 1 to 32 and ∼5-fold lower than the rate for the PVR-binding sites. Only VP1 143-Ile, a non-NAg site surface residue and known attenuation site, appeared to be under strong negative selection.IMPORTANCE The high rate of poliovirus evolution is offset by strong selection against amino acid replacement at most positions of the capsid. Consequently, poliovirus vaccines developed from strains isolated decades ago have been used worldwide to bring wild polioviruses almost to extinction. The apparent antigenic stability of poliovirus obscures a dynamic of continuous change within the neutralizing antigenic (NAg) sites. During 7 years of a large outbreak in Nigeria, the circulating type 2 vaccine-derived polioviruses generated 120 different NAg site variants via multiple independent pathways. Nonetheless, overall antigenic evolution was constrained, as no isolate had fixed more than 5 amino acid differences from the Sabin 2 NAg sites, and the most divergent isolates were efficiently neutralized by human immune sera. Evolution elsewhere in the capsid was also constrained. Amino acids binding the poliovirus receptor were strongly conserved, and extensive variation in the VP1 amino terminus still conserved a predicted amphipathic helix.

Construction of a recombinant baculovirus expressing swine hepatitis E Virus ORF2 and preliminary research on its immune effect.

In the swine hepatitis E virus (HEV), open reading frame 2 (ORF2) is rich in antigenic determinants and neutralizing epitopes that could induce immune protection. We chose the Bac-to-Bac® Baculovirus Expression System to express fragments containing the critical neutralizing antigenic sites within the HEV ORF2 protein of pigs to obtain a recombinant baculovirus. The fragment of swine HEV ORF2 region (1198-1881bp) was cloned into vector pFastBacTM. A recombinant baculovirus, rBacmid-ORF2, was obtained after transposition and transfection. The molecular mass of the recombinant protein was 26 kDa. Mice were immunized by the intraperitoneal and oral routes with cell lysates of recombinant baculovirus rBacmid-ORF2. Serum and feces of the mice were collected separately at 0, 14, 28, and 42 d after immunization and the antibody levels of IgG and secretory IgA against swine HEV were determined using an enzyme-linked immunosorbent assay. The results suggested that rBacmid-ORF2 induced antibodies of the humoral and mucosal immune responses in mice and that the oral route was significantly superior to the intraperitoneal route. This is the first study to demonstrate that that recombinant baculovirus swine HEV ORF2 could induce humoral and mucosal immune responses in mice.

Characterization of a novel VIIl sub-genotype of Newcastle disease virus circulating in Iran

ABSTRACTNewcastle disease is an economically important and highly contagious disease affecting wild and domestic avian species. Despite extensive vaccination efforts within the poultry industry, Newcastle disease virus (NDV) outbreaks causing significant economic losses still occur. Rural chickens may act as a potential reservoir of NDVs for commercial poultry due to poor biosecurity and inadequate vaccination. The aim of this study was to investigate the phylogenetic relationship and molecular characterization of eight NDVs isolated from backyard poultry in Iran during 2011–2013. The complete coding sequence of fusion (F) and haemagglutinin-neuraminidase (HN) genes of eight NDVs were determined and compared with other published NDVs. Based on inter-population distances and phylogenetic topology between available NDV categories, Iranian isolates formed a novel VIIl sub-genotype distinct from previous groups designated in genotype VII. Furthermore, both F and HN genes of the Iranian isolates shared high nucleotide sequence similarity with viruses isolated in China. All viruses analysed contained a polybasic cleavage site motif (111G/RRRQKR↓F117), indicating that all isolates could be categorized as a virulent pathotype. No mutation was observed in the neutralizing epitopes of the F protein. Analysis of amino acids associated with neutralizing antigenic sites within the HN protein revealed that all isolates exhibited a unique amino acid (Q) at position 347. These results emphasize the need for strengthening the biosecurity measures implemented on village flocks and practicing a mandatory vaccination programme for local poultry. Moreover, continuous monitoring of NDVs in different species of birds can help to gain more knowledge about the evolution of this virus and prevent future panzootics.

Immunogenicity of T7 bacteriophage nanoparticles displaying G-H loop of foot-and-mouth disease virus (FMDV)

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes severe economic losses worldwide. The G-H loop of the FMDV VP1 structural protein is the major neutralizing antigenic site. However, a fully protective G-H loop peptide vaccine requires the addition of promiscuous Th sites from a source outside VP1. Thus, we demonstrated the potential of T7 bacteriophage based nanoparticles displaying a genetically fused G-H loop peptide (T7-GH) as a FMDV vaccine candidate. Recombinant T7-GH phage was constructed by inserting the G-H loop coding region into the T7 Select 415-1b vector. Purified T7-GH phage nanoparticles were analyzed by SDS-PAGE, Western blot and Dot-ELISA. Pigs seronegative for FMDV exposure were immunized with T7-GH nanoparticles along with the adjuvant Montanide ISA206, and two commercially available FMDV vaccines (InactVac and PepVac). Humoral and cellular immune responses, as well as protection against virulent homologous virus challenge were assessed following single dose immunization. Pigs immunized T7-GH developed comparable anti-VP1 antibody titers to PepVac, although lower LPBE titers than was induced by InactVac. Antigen specific lymphocyte proliferation was detected in T7-GH group similar to that of PepVac group, however, weaker than InactVac group. Pigs immunized with T7-GH developed a neutralizing antibody response stronger than PepVac, but weaker than InactVac. Furthermore, 80% (4/5) of T7-GH immunized pigs were protected from challenge with virulent homologous virus. These findings demonstrate that the T7-GH phage nanoparticles were effective in eliciting antigen specific immune responses in pigs, highlighting the value of such an approach in the research and development of FMDV vaccines.

Identification of novel E347Q and G362K amino acid substitutions in HN neutralization epitope and major antigenic difference in novel sub-genotype VIIj isolates.

Hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in induction of immune response against Newcastle disease infection. Mutation in residues 347 and 362 of HN linear antigenic site has been identified to be responsible for antigenic variations. Hence we studied antigenic difference between sub-genotype VIIj isolates and vaccine strains by the use of polyclonal serum against LaSota strain in hemagglutination inhibition test. Furthermore, epitope patterns of the isolates under study were analyzed and compared to HN sequences in GenBank. The results demonstrated that new Newcastle disease isolates (Mazandaran and Behshahr) had hemagglutination inhibition (HI) titer three and five, respectively while LaSota strain titer was eight. In addition, observation of sequences and epitope patterns revealed three unique amino acid substitutions (D144N, E347Q and G362K) in HN protein. E347Q and G362K mutations were located in neutralization antigenic site. Thus, we suggest that, these two novel amino acid substitutions in major linear epitope might be responsible for antigenic variation and decrease of HI activity.

Role of asparagine at position 562 in dimerization and immunogenicity of the hepatitis E virus capsid protein

The hepatitis E virus (HEV) capsid protein, pORF2, contains 2 potential N-glycosylation sites, N137 and N310, located in the S domain, and one site, N562, in the P domain. The last domain located at positions 454-606 aa forms a protruding spike from the shell, with N562 being located in the apical center of the spike, which is also a cell-attachment region and neutralizing antigenic site. Here, we expressed in Pichia pastoris a recombinant polypeptide p179 comprising the region of 439-617 aa of the HEV pORF2 as well as a set of 4 mutant proteins containing substitutions of Q, D, P and Y instead of N at position 562. All proteins were shown to be secreted from yeast. Using SDS-PAGE, Western blot analysis and tunicamycin treatment assay, we showed that the wild-type (wt) protein, p179N562, and 2 mutant variants, p179N562Q and p179N562D, formed homodimers but only the wt protein was shown to be glycosylated. As homodimers, all 3 proteins were immunoreactive with a neutralizing monoclonal antibody (5G5); however, they did not immunoreact with 5G5 after denaturation into monomers. Two other mutant variants, p179N562P and p179N562Y, did not form homodimers but were immunoreactive with the 5G5 antibody. The wt protein was shown to be less immunoreactive with 5G5 than the mutant variants in a double-antibody sandwich ELISA, suggesting a role of glycosylation at N562 in reducing antibody binding. In vitro neutralization experiments showed a more efficient neutralization with mouse antibody against p179N562P and p179N562Y than against the other 3 proteins. These findings indicate that specific substitutions at position 562 have a more measurable effect on the activity of the HEV neutralizing epitope than dimerization or glycosylation of the structural protein. Furthermore, the secretion of monomers fully immunoreactive may call into question the importance of dimerization for an effective presentation of HEV neutralization epitopes.

Structural basis for the antibody neutralization of herpes simplex virus.

Glycoprotein D (gD) of herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317 Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD-nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.