Computational identification of HCV neutralizing antibodies with a common HCDR3 disulfide bond motif in the antibody repertoires of infected individuals.

Despite recent success in hepatitis C virus (HCV) treatment using antivirals, an HCV vaccine is still needed to prevent reinfections in treated patients, to avert the emergence of drug-resistant strains, and to provide protection for people with no access to the antiviral therapeutics. The early production of broadly neutralizing antibodies (bNAbs) associates with HCV clearance. Several potent bNAbs bind a conserved HCV glycoprotein E2 epitope using an unusual heavy chain complementarity determining region 3 (HCDR3) containing an intra-loop disulfide bond. Isolation of additional structurally-homologous bNAbs would facilitate the recognition of key determinants of such bNAbs and guide rational vaccine design. Here we report the identification of new antibodies containing an HCDR3 disulfide bond motif using computational screening with the Rosetta software. Using the newly-discovered and already-known members of this antibody family, we review the required HCDR3 amino acid composition and propose determinants for the bent versus straight HCDR3 loop conformation observed in these antibodies.

Cross-Protection against MERS-CoV by Prime-Boost Vaccination Using Viral Spike DNA and Protein.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness and has a high mortality of ∼34%. However, since its discovery in 2012, an effective vaccine has not been developed for it. To develop a vaccine against multiple strains of MERS-CoV, we targeted spike glycoprotein (S) using prime-boost vaccination with DNA and insect cell-expressed recombinant proteins for the receptor-binding domain (RBD), S1, S2, SΔTM, or SΔER. Our S subunits were generated using an S sequence derived from the MERS-CoV EMC/2012 strain. We examined humoral and cellular immune responses of various combinations with DNA plasmids and recombinant proteins in mice. Mouse sera immunized with SΔER DNA priming/SΔTM protein boosting showed cross-neutralization against 15 variants of S-pseudovirions and the wild-type KOR/KNIH/002 strain. In addition, these immunizations provided full protection against the KOR/KNIH/002 strain challenge in human DPP4 knock-in mice. These findings suggest that vaccination with the S subunits derived from one viral strain can provide cross-protection against variant MERS-CoV strains with mutations in S. DNA priming/protein boosting increased gamma interferon production, while protein-alone immunization did not. The RBD subunit alone was insufficient to induce neutralizing antibodies, suggesting the importance of structural conformation. In conclusion, heterologous DNA priming with protein boosting is an effective way to induce both neutralizing antibodies and cell-mediated immune responses for MERS-CoV vaccine development. This study suggests a strategy for selecting a suitable platform for developing vaccines against MERS-CoV or other emerging coronaviruses.IMPORTANCE Coronavirus is an RNA virus with a higher mutation rate than DNA viruses. Therefore, a mutation in S-protein, which mediates viral infection by binding to a human cellular receptor, is expected to cause difficulties in vaccine development. Given that DNA-protein vaccines promote stronger cell-mediated immune responses than protein-only vaccination, we immunized mice with various combinations of DNA priming and protein boosting using the S-subunit sequences of the MERS-CoV EMC/2012 strain. We demonstrated a cross-protective effect against wild-type KOR/KNIH/002, a strain with two mutations in the S amino acids, including one in its RBD. The vaccine also provided cross-neutralization against 15 different S-pseudotyped viruses. These suggested that a vaccine targeting one variant of S can provide cross-protection against multiple viral strains with mutations in S. The regimen of DNA priming/Protein boosting can be applied to the development of other coronavirus vaccines.

An ultralong CDRH2 in HCV neutralizing antibody demonstrates structural plasticity of antibodies against E2 glycoprotein.

A vaccine protective against diverse HCV variants is needed to control the HCV epidemic. Structures of E2 complexes with front layer-specific broadly neutralizing antibodies (bNAbs) isolated from HCV-infected individuals, revealed a disulfide bond-containing CDRH3 that adopts straight (individuals who clear infection) or bent (individuals with chronic infection) conformation. To investigate whether a straight versus bent disulfide bond-containing CDRH3 is specific to particular HCV-infected individuals, we solved a crystal structure of the HCV E2 ectodomain in complex with AR3X, a bNAb with an unusually long CDRH2 that was isolated from the chronically-infected individual from whom the bent CDRH3 bNAbs were derived. The structure revealed that AR3X utilizes both its ultralong CDRH2 and a disulfide motif-containing straight CDRH3 to recognize the E2 front layer. These results demonstrate that both the straight and bent CDRH3 classes of HCV bNAb can be elicited in a single individual, revealing a structural plasticity of VH1-69-derived bNAbs.

Characterization of novel monoclonal antibodies against MERS-coronavirus spike protein.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe pulmonary infection, with ∼35 % mortality. Spike glycoprotein (S) of MERS-CoV is a key target for vaccines and therapeutics because S mediates viral entry and membrane-fusion to host cells. Here, four different S subunit proteins, receptor-binding domain (RBD; 358-606 aa), S1 (1-751 aa), S2 (752-1296 aa), and SΔTM (1-1296 aa), were generated using the baculoviral system and immunized in mice to develop neutralizing antibodies. We developed 77 hybridomas and selected five neutralizing mAbs by immunization with SΔTM against MERS-CoV EMC/2012 strain S-pseudotyped lentivirus. However, all five monoclonal antibodies (mAb) did not neutralize the pseudotyped V534A mutation. Additionally, one mAb RBD-14F8 did not show neutralizing activity against pseudoviruses with amino acid substitution of L506 F or D509 G (England1 strain, EMC/2012 L506 F, and EMC/2012 D509 G), and RBD-43E4 mAb could not neutralize the pseudotyped I529 T mutation, while three other neutralizing mAbs showed broad neutralizing activity. This implies that the mutation in residue 506-509, 529, and 534 of S is critical to generate neutralization escape variants of MERS-CoV. Interestingly, all five neutralizing mAbs have binding affinity to RBD, although most mAbs generated by RBD did not have neutralizing activity. Additionally, chimeric antibodies of RBD-14F8 and RBD-43E4 with human Fc and light chain showed neutralizing effect against wild type MERS-CoV KOR/KNIH/002, similar to the original mouse mAbs. Thus, our mAbs can be utilized for the identification of specific mutations of MERS-CoV.

Cross-Protective Shigella Whole-Cell Vaccine With a Truncated O-Polysaccharide Chain.

Shigella is a highly prevalent bacterium causing acute diarrhea and dysentery in developing countries. Shigella infections are treated with antibiotics but Shigellae are increasingly resistant to these drugs. Vaccination can be a countermeasure against emerging antibiotic-resistant shigellosis. Because of the structural variability in Shigellae O-antigen polysaccharides (Oag), cross-protective Shigella vaccines cannot be derived from single serotype-specific Oag. We created an attenuated Shigella flexneri 2a strain with one rather than multiple Oag units by disrupting the Oag polymerase gene (Δwzy), which broadened protective immunogenicity by exposing conserved surface proteins. Inactivated Δwzy mutant cells combined with Escherichia coli double mutant LT(R192G/L211A) as adjuvant, induced potent antibody responses to outer membrane protein PSSP-1, and type III secretion system proteins IpaB and IpaC. Intranasal immunization with the vaccine preparation elicited cross-protective immunity against S. flexneri 2a, S. flexneri 3a, S. flexneri 6, and Shigella sonnei in a mouse pneumonia model. Thus, S. flexneri 2a Δwzy represents a promising candidate strain for a universal Shigella vaccine.

Immunogenicity of a bivalent killed thimerosal-free oral cholera vaccine, Euvichol, in an animal model.

PURPOSE: An oral cholera vaccine (OCV), Euvichol, with thimerosal (TM) as preservative, was prequalified by the World Health Organization (WHO) in 2015. In recent years, public health services and regulatory bodies recommended to eliminate TM in vaccines due to theoretical safety concerns. In this study, we examined whether TM-free Euvichol induces comparable immunogenicity to its TM-containing formulation in animal model. MATERIALS AND METHODS: To evaluate and compare the immunogenicity of the two variations of OCV, mice were immunized with TM-free or TM-containing Euvichol twice at 2-week interval by intranasal or oral route. One week after the last immunization, mice were challenged with Vibrio cholerae O1 and daily monitored to examine the protective immunity against cholera infection. In addition, serum samples were obtained from mice to measure vibriocidal activity and vaccine-specific IgG, IgM, and IgA antibodies using vibriocidal assay and enzyme-linked immunosorbent assay, respectively. RESULTS: No significant difference in immunogenicity, including vibriocidal activity and vaccine-specific IgG, IgM, and IgA in serum, was observed between mice groups administered with TM-free and -containing Euvichol, regardless of immunization route. However, intranasally immunized mice elicited higher levels of serum antibodies than those immunized via oral route. Moreover, intranasal immunization completely protected mice against V. cholerae challenge but not oral immunization. There was no significant difference in protection between two Euvichol variations. CONCLUSION: These results suggested that TM-free Euvichol could provide comparable immunogenicity to the WHO prequalified Euvichol containing TM as it was later confirmed in a clinical study. The pulmonary mouse cholera model can be considered useful to examine in vivo the potency of OCVs.

Protein energy malnutrition alters mucosal IgA responses and reduces mucosal vaccine efficacy in mice.

Oral vaccine responsiveness is often lower in children from less developed countries. Childhood malnutrition may be associated with poor immune response to oral vaccines. The present study was designed to investigate whether protein energy malnutrition (PEM) impairs B cell immunity and ultimately reduces oral vaccine efficacy in a mouse model. Purified isocaloric diets containing low protein (1/10 the protein of the control diet) were used to determine the effect of PEM. PEM increased both nonspecific total IgA and oral antigen-specific IgA in serum without alteration of gut permeability. However, PEM decreased oral antigen-specific IgA in feces, which is consistent with decreased expression of polymeric Immunoglobulin receptor (pIgR) in the small intestine. Of note, polymeric IgA was predominant in serum under PEM. In addition, PEM altered B cell development status in the bone marrow and increased the frequency of IgA-secreting B cells, as well as IgA secretion by long-lived plasma cells in the small intestinal lamina propria. Moreover, PEM reduced the protective efficacy of the mucosally administered cholera vaccine and recombinant attenuated Salmonella enterica serovar Typhimurium vaccine in a mouse model. Our results suggest that PEM can impair mucosal immunity where IgA plays an important role in host protection and may partly explain the reduced efficacy of oral vaccines in malnourished subjects.

Natural killer T cell sensitization during neonatal respiratory syncytial virus infection induces eosinophilic lung disease in re-infected adult mice.

Respiratory syncytial virus (RSV) is a major viral pathogen that causes severe lower respiratory tract infections in infants and the elderly worldwide. Infants with severe RSV bronchiolitis tend to experience more wheezing and asthma in later childhood. Because invariant natural killer T (iNKT) cells are associated with the asthma pathology, we investigated whether neonatal iNKT cells are involved in the aggravation of pulmonary diseases following RSV infection in mice. Intranasal exposure to the iNKT cell ligand α-galactosylceramide (α-GC) with RSV primary infection in neonatal mice elicited neither cytokine production (except for a slight increase of IL-5) nor pulmonary eosinophilia, despite the presence of both CD1d+ cells and NKT cells. Interestingly, in adult mice re-infected with RSV, neonatal iNKT cell sensitization by α-GC during RSV primary infection resulted in much higher levels of pulmonary Th2 cytokines and elevated eosinophilia with airway hyperresponsiveness, whereas this was not observed in cd1d knockout mice. In contrast, α-GC priming of adults during RSV re-infection did not induce more severe airway symptoms than RSV re-infection in the absence of α-GC. α-GC co-administration during RSV primary infection facilitated RSV clearance regardless of age, but viral clearance following re-infection was not iNKT cell-dependent. This study clearly demonstrates that RSV-induced immune responses can be altered by iNKT cells, suggesting that neonatal iNKT cell sensitization during RSV primary infection is associated with exacerbation of pulmonary diseases following RSV re-infection in adulthood.

Shigella outer membrane protein PSSP-1 is broadly protective against Shigella infection.

In developing countries, Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella.

Neonatal immunization with respiratory syncytial virus glycoprotein fragment induces protective immunity in the presence of maternal antibodies in mice.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly worldwide. The significant morbidity and mortality associated with this infection underscores the urgent need for development of RSV vaccine. In this study, we first show that intranasal administration of RSV glycoprotein core fragment (Gcf) to neonatal mice can induce systemic humoral immune responses and protective immunity against RSV without causing lung eosinophilia, although antibody response was shifted to a Th2 response. Next, we examined whether the presence of maternal anti-RSV antibodies would affect the responsiveness and protection efficacy of Gcf in newborn mice, since infants can possess RSV-specific maternal antibodies due to frequent RSV re-infections to adults. Intranasal administration of Gcf induced antibody response and increased IFNγ secretion and protected mice against RSV challenge without severe lung eosinophilia, even in the presence of high levels of RSV-specific maternal antibodies. Thus, our findings suggest that Gcf may be an effective and safe RSV vaccine during the neonatal period.

Identification of reassortant pandemic H1N1 influenza virus in Korean pigs.

Since the 2009 pandemic human H1N1 influenza A virus emerged in April 2009, novel reassortant strains have been identified throughout the world. This paper describes the detection and isolation of reassortant strains associated with human pandemic influenza H1N1 and swine influenza H1N2 (SIV) viruses in swine populations in South Korea. Two influenza H1N2 reassortants were detected, and subtyped by PCR. The strains were isolated using Madin- Darby canine kidney (MDCK) cells, and genetically characterized by phylogenetic analysis for genetic diversity. They consisted of human, avian, and swine virus genes that were originated from the 2009 pandemic H1N1 virus and a neuraminidase (NA) gene from H1N2 SIV previously isolated in North America. This identification of reassortment events in swine farms raises concern that reassortant strains may continuously circulate within swine populations, calling for the further study and surveillance of pandemic H1N1 among swine.

Population dynamics and ORF3 gene evolution of porcine circovirus type 2 circulating in Korea.

This study was conducted to investigate the status and population dynamics of porcine circovirus type 2(PCV2) in Korea and to assess the molecular evolutionary pattern of the two biologically important, overlapping open reading frames, the ORF1 and ORF3 genes. A wide range of PCV2 genomic sequences (entire genome, ORF1, ORF2 and ORF3) collected between 2001 and 2010 were analyzed using the Bayesian Markov chain Monte Carlo and maximum-likelihood approaches. These techniques identified the PCV2d genotype and the 2Ek cluster of PCV2a in Korea for the first time. Second, the genotypic shift of PCV2b dominating over PCV2a likely occurred between 2002 and 2004 due to a population expansion of PCV2b. In the context of positive Darwinian selection, the results uncovered independent evolutionary patterns in the ORF3 gene compared to the overlapping ORF1 gene and new sites in the viral ORFs/proteins that might relate to differences in the biological properties of the PCV2 groups.

Effect of maternal immune status on responsiveness of bacillus calmette-gurin vaccination in mouse neonates.

OBJECTIVES: Bacillus Calmette-Guérin (BCG) vaccination has proven to be efficient in immunologically naïve infants; however, it has not been investigated that maternal natural exposure to Mycobacterium and/or BCG vaccine could influence the characteristics of immune responses to BCG in newborns. In this study, we analyzed whether the maternal immune status to M tuberculosis (M tb) can affect neonatal immunity to BCG using a mouse model. METHODS: Neonates were obtained from mice that were previously exposed to live BCG, to live M avium, or to heat-killed M tb H37Rv, and from naïve control mothers. One week after birth, the neonates were divided into two subgroups: one group immunized with live BCG via the subcutaneous route and the other group of neonates sham-treated. Interferon-gamma (IFNγ) secretion in response to in vitro stimulation with heat-killed BCG or purified protein derivative (PPD) was examined. Protection against M tb infection was evaluated by challenging mice nasally with live M tb H37Rv followed by counting colonies from spleen and lung homogenates. RESULTS: BCG-immunized neonates showed increased IFNγ secretion in response to heat-killed BCG or PPD. All mice in BCG-immunized neonates subgroups showed reduced bacterial burden (colony forming unit) in the lungs when compared with control naive neonate mice. However, no statistically significant difference was observed when comparing BCG-immunized mice born from mothers previously exposed to M avium or immunized with either heat-killed H37Rv or live BCG and mice born from naïve mothers. CONCLUSION: The maternal immune status to M tb does not appear to impact on the immunogenicity of BCG vaccine in their progeny in our experimental conditions.

Detection and genetic analysis of porcine hemagglutinating encephalomyelitis virus in South Korea.

Porcine hemagglutinating encephalomyelitis virus (PHEV) causes vomiting and wasting disease (VWD) or encephalomyelitis, and primarily affects pigs under 3 weeks of age. In this study, we detected PHEV from clinically ill pigs in conventional pig farms in South Korea. From November 2009 to March 2010, a total of 239 pig tissue samples from 91 farms were tested by nested RT-PCR. Among 239 samples, 22 samples from 17 farms were positive for PHEV. The detection rate of suckling pigs, weaning pigs, growers and finishers were 14.3% (12/84), 6.5% (7/107), 7% (3/43), and 0% (0/5), respectively. Symptoms were neurological, respiratory, enteric sign (diarrhea), or nasal bleeding. All pigs were co-infected with other viruses and bacteria and this might have resulted in age variation and clinical signs in the affected pigs. Phylogenetic analysis showed that the PHEV-positive samples and PHEV reference strains were clustered in the same group. These findings imply the presence of only one genogroup of PHEV, regardless of porcine age, clinical signs, and geographical location.

Molecular detection of porcine kobuviruses in pigs in Korea and their association with diarrhea.

Kobuviruses are small, non-enveloped viruses with a single-stranded, positive-sense genomic RNA, belonging to the family Picornaviridae, a highly diverse family of important pathogens of human and other animals. Porcine kobuvirus has been found recently, and consequently, information about the virus is lacking. In this study, we identified porcine kobuviruses from pigs in Korea by RT-PCR, cloning and sequencing, and we showed the existence of genetic diversity among geographically separated porcine kobuviruses through genetic and phylogenetic analysis. Epidemiological studies of porcine kobuvirus linked to diarrhea indicated that porcine kobuvirus infections are endemic in diarrheic pigs in Korea. Statistical analysis of the porcine kobuvirus positive rate between diarrheic and healthy pigs as well as a survey for other enteric pathogens in diarrheic pigs suggests that porcine kobuvirus may play a role as a causative agent of gastroenteritis in pigs.

Simultaneous detection and subtyping of porcine endogenous retroviruses proviral DNA using the dual priming oligonucleotide system.

The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes.

Porcine noroviruses and sapoviruses on Korean swine farms.

Porcine noroviruses (NoVs) and sapoviruses (SaVs), which belong to the family Caliciviridae, have been considered potential zoonotic agents for human infection, and several cases have been reported in Asian countries. In this study, a total of 537 porcine fecal samples collected from 64 swine farms in Korea were tested. Among 537 samples, porcine NoVs were detected by semi-nested RT-PCR in ten samples (1.9%), and porcine SaVs were detected by RT-PCR in 60 samples (11.2%), showing their circulation in Korea. The porcine NoVs were genetically related to strains of genotypes 11 and 18, of genogroup II (GII) of the genus Norovirus. The porcine SaV strains were genetically related to the porcine enteric calicivirus Cowden strain and to the previously identified Korean porcine strains in genogroup III (GIII) of the genus Sapovirus. In no case was co-infection with both NoV and SaV observed in one pig. This is the first report describing porcine NoVs identified in Korea.

Phylogenetic and recombination analysis of genomic sequences of PCV2 isolated in Korea.

The complete genomic sequences of 13 PCV2 viruses obtained between 2005 and 2007 were analyzed in order to determine their phylogenetic relationship and identify possible recombination events between PCV2a and PCV2b. Twelve PCV2b viruses and one PCV2a virus were identified by phylogenetic analysis. Notably, two PCV2b viruses (PF163 and C7201-1) were shown to belong to the 1B subgroup of PCV2b, which had not been previously reported in Korea. Theses two viruses were also predicted to be possible recombinants between PCV2a (the minor parent) and PCV2b (the major parent) by the RDP program (P < 0.01). A recombination site was predicted to exist in ORF1 of both viruses. This additional evidence of PCV2 recombination in Korea further supports the important role of recombination in genetic evolution.