Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

Background

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear.

Methods/Findings

In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment.

Conclusions

Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.     

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

Background

The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

Methodology/Principal Findings

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

Conclusion/Significance

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.     

Exogenous interleukin-6, interleukin-13, and interferon-gamma provoke pulmonary abnormality with mild edema in enterovirus 71-infected mice

Background

Neonatal mice developed neurological disease and pulmonary dysfunction after an infection with a mouse-adapted human Enterovirus 71 (EV71) strain MP4. However, the hallmark of severe human EV71 infection, pulmonary edema (PE), was not evident.

Methods

To test whether EV71-induced PE required a proinflammatory cytokine response, exogenous pro-inflammatory cytokines were administered to EV71-infected mice during the late stage of infection.

Results

After intracranial infection of EV71/MP4, 7-day-old mice developed hind-limb paralysis, pulmonary dysfunction, and emphysema. A transient increase was observed in serum IL-6, IL-10, IL-13, and IFN-γ, but not noradrenaline. At day 3 post infection, treatment with IL-6, IL-13, and IFN-γ provoked mild PE and severe emphysema that were accompanied by pulmonary dysfunction in EV71-infected, but not herpes simplex virus-1 (HSV-1)-infected control mice. Adult mice did not develop PE after an intracerebral microinjection of EV71 into the nucleus tractus solitarii (NTS). While viral antigen accumulated in the ventral medulla and the NTS of intracerebrally injected mice, neuronal loss was observed in the ventral medulla only.

Conclusions

Exogenous IL-6, IL-13, and IFN-γ treatment could induce mild PE and exacerbate pulmonary abnormality of EV71-infected mice. However, other factors such as over-activation of the sympathetic nervous system may also be required for the development of classic PE symptoms.     

Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection

Background

Enterovirus 71 (EV71) is the main causative agent of Hand, Foot and Mouth disease (HFMD) and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment.

Methodology/Principal Finding

In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215–219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16).

Conclusion

We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.     

The Interplays between Autophagy and Apoptosis Induced by Enterovirus 71

Background

Enterovirus 71 (EV71) is the causative agent of human diseases with distinct severity, from mild hand, foot and mouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of viral pathogenesis as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. EV71 virus induces autophagy and apoptosis; however, the relationship between EV71-induced autophagy and apoptosis as well as the influence of autophagy and apoptosis on virus virulence remains unclear.

Methodology/Principal Findings

In this study, it was observed that the Anhui strain of EV71 induced autophagy and apoptosis in human rhabdomyosarcoma (RD-A) cells. Additionally, by either applying chemical inhibitors or knocking down single essential autophagic or apoptotic genes, inhibition of EV71 induced autophagy inhibited the apoptosis both at the autophagosome formation stage and autophagy execution stage. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion promoted apoptosis. In reverse, the inhibition of EV71-induced apoptosis contributed to the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II and degradation of sequestosome 1 (SQSTM1/P62). Furthermore, the inhibition of autophagy in the autophagsome formation stage or apoptosis decreased the release of EV71 viral particles.

Conclusions/Significance

In conclusion, the results of this study not only revealed novel aspect of the interplay between autophagy and apoptosis in EV71 infection, but also provided a new insight to control EV71 infection.     

Avirulent Marek’s Disease Virus Type 1 Strain 814 Vectored Vaccine Expressing Avian Influenza (AI) Virus H5 Haemagglutinin Induced Better Protection Than Turkey Herpesvirus Vectored AI Vaccine

Background

Herpesvirus of turkey (HVT) as a vector to express the haemagglutinin (HA) of avian influenza virus (AIV) H5 was developed and its protection against lethal Marek’s disease virus (MDV) and highly pathogenic AIV (HPAIV) challenges was evaluated previously. It is well-known that avirulemt MDV type 1 vaccines are more effective than HVT in prevention of lethal MDV infection. To further increase protective efficacy against HPAIV and lethal MDV, a recombinant MDV type 1 strain 814 was developed to express HA gene of HPAIV H5N1.

Methodology/Principal Findings

A recombinant MDV-1 strain 814 expressing HA gene of HPAIV H5N1 virus A/goose/Guangdong/3/96 at the US2 site (rMDV-HA) was developed under the control of a human CMV immediate-early promoter. The HA expression in the rMDV-HA was tested by immunofluorescence and Western blot analyses, and in vitro and in vivo growth properties of rMDV-HA were also analyzed. Furthermore, we evaluated and compared the protective immunity of rMDV-HA and previously constructed rHVT-HA against HPAIV and lethal MDV. Vaccination of chickens with rMDV-HA induced 80% protection against HPAIV, which was better than the protection rate by rHVT-HA (66.7%). In the animal study with MDV challenge, chickens immunized with rMDV-HA were completely protected against virulent MDV strain J-1 whereas rHVT-HA only induced 80% protection with the same challenge dose.

Conclusions/Significance

The rMDV-HA vaccine was more effective than rHVT-HA vaccine for protection against lethal MDV and HPAIV challenges. Therefore, avirulent MDV type 1 vaccine is a better vector than HVT for development of a recombinant live virus vaccine against virulent MDV and HPAIV in poultry.     

Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model

Background

We previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.

Results

We demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH₄Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.

Conclusions

In this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.     

Heterosubtypic antibody response elicited with seasonal influenza vaccine correlates partial protection against highly pathogenic H5N1 virus

Background

The spread of highly pathogenic avian influenza (HPAI) H5N1 virus in human remains a global health concern. Heterosubtypic antibody response between seasonal influenza vaccine and potential pandemic influenza virus has important implications for public health. Previous studies by Corti et al. and by Gioia et al. demonstrate that heterosubtypic neutralizing antibodies against the highly pathogenic H5N1 virus can be elicited with a seasonal influenza vaccine in humans. However, whether such response offers immune protection against highly pathogenic H5N1 virus remained to be determined.

Methodology/Principal Findings

In this study, using a sensitive influenza HA (hemagglutinin) and NA (neuraminidase) pseudotype-based neutralization (PN) assay we first confirmed that low levels of heterosubtypic neutralizing antibody response against H5N1 virus were indeed elicited with seasonal influenza vaccine in humans. We then immunized mice with the seasonal influenza vaccine and challenged them with lethal doses of highly pathogenic H5N1 virus. As controls, we immunized mice with homosubtypic H5N1 virus like particles (VLP) or PBS and challenged them with the same H5N1 virus. Here we show that low levels of heterosubtypic neutralizing antibody response were elicited with seasonal influenza vaccine in mice, which were significantly higher than those in PBS control. Among them 2 out of 27 whose immune sera exhibited similar levels of neutralizing antibody response as VLP controls actually survived from highly pathogenic H5N1 virus challenge.

Conclusions/Significance

Therefore, we conclude that low levels of heterosubtypic neutralizing antibody response are indeed elicited with seasonal influenza vaccine in humans and mice and at certain levels such response offers immune protection against severity of H5N1 virus infection.     

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates

Background

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial.

Principal Finding

In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7–10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30–43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37°C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4°C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates.

Conclusion

These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.     

Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system

Background

Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.

Principal Finding

In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >10⁶ TCID₅₀/mL by 6 days post infection when a MOI of 10⁻⁵ was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225).

Conclusion

These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.     

Comparative Analysis of the Immunogenicity and Protective Effects of Inactivated EV71 Vaccines in Mice

Background

Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD). Three inactivated EV71 whole-virus vaccines of different strains developed by different manufacturers in mainland China have recently entered clinical trials. Although several studies on these vaccines have been published, a study directly comparing the immunogenicity and protective effects among them has not been carried out, which makes evaluating their relative effectiveness difficult. Thus, properly comparing newly developed vaccines has become a priority, especially in China.

Methods and Findings

This comparative immunogenicity study was carried out on vaccine strains (both live and inactivated), final container products (FCPs) without adjuvant, and corresponding FCPs containing adjuvant (FCP-As) produced by three manufacturers. These vaccines were evaluated by neutralizing antibody (NAb) responses induced by the same or different dosages at one or multiple time points post-immunization. The protective efficacy of the three vaccines was also determined in one-day-old ICR mice born to immunized female mice. Survival rates were observed in these suckling mice after challenge with 20 LD₅₀ of EV71/048M3C2. Three FCP-As, in a dose of 200 U, generated nearly 100% NAb positivity rates and similar geometric mean titers (GMTs), especially at 14–21 days post-inoculation. However, the dynamic NAb responses were different among three vaccine strains or three FCPs. The FCP-As at the lowest dose used in clinical trials (162 U) showed good protective effects in suckling mice against lethal challenge (90–100% survival), while the ED₅₀ of NAb responses and protective effects varied among three FCP-As.

Conclusions

These studies establish a standard method for measuring the immunogenicity of EV71 vaccines in mice. The data generated from our mouse model study indicated a clear dose-response relationship, which is important for vaccine quality control and assessment, especially for predicting protective efficacy in humans when combined with future clinical trial results.     

A Single Immunization with MVA Expressing GnGc Glycoproteins Promotes Epitope-specific CD8+-T Cell Activation and Protects Immune-competent Mice against a Lethal RVFV Infection

Background

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen causing an important disease in ruminants often transmitted to humans after epizootic outbreaks in African and Arabian countries. To help combat the spread of the disease, prophylactic measures need to be developed and/or improved.

Methodology/Principal Findings

In this work, we evaluated the immunogenicity and protective efficacy of recombinant plasmid DNA and modified vaccinia virus Ankara (rMVA) vectored vaccines against Rift Valley fever in mice. These recombinant vaccines encoded either of two components of the Rift Valley fever virus: the viral glycoproteins (Gn/Gc) or the nucleoprotein (N). Following lethal challenge with live RVFV, mice immunized with a single dose of the rMVA-Gn/Gc vaccine showed no viraemia or clinical manifestation of disease, but mounted RVFV neutralizing antibodies and glycoprotein specific CD8+ T-cell responses. Neither DNA-Gn/Gc alone nor a heterologous prime-boost immunization schedule (DNA-Gn/Gc followed by rMVAGn/Gc) was better than the single rMVA-Gn/Gc immunization schedule with regards to protective efficacy. However, the rMVA-Gn/Gc vaccine failed to protect IFNAR^−/− mice upon lethal RVFV challenge suggesting a role for innate responses in protection against RVFV. Despite induction of high titer antibodies against the RVFV nucleoprotein, the rMVA-N vaccine, whether in homologous or heterologous prime-boost schedules with the corresponding recombinant DNA vaccine, only conferred partial protection to RVFV challenge.

Conclusions/Significance

Given the excellent safety profile of rMVA based vaccines in humans and animals, our data supports further development of rMVA-Gn/Gc as a vaccine strategy that can be used for the prevention of Rift Valley fever in both humans and livestock.     

H5N1 whole-virus vaccine induces neutralizing antibodies in humans which are protective in a mouse passive transfer model

Background

Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge.

Methods

We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus.

Results

Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species.

Conclusions

These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines.     

Mutations in the non-structural protein region contribute to intra-genotypic evolution of enterovirus 71

Background

Clinical manifestations of enterovirus 71 (EV71) range from herpangina, hand-foot-and-mouth disease (HFMD), to severe neurological complications. Unlike the situation of switching genotypes seen in EV71 outbreaks during 1998–2008 in Taiwan, genotype B5 was responsible for two large outbreaks in 2008 and 2012, respectively. In China, by contrast, EV71 often persists as a single genotype in the population and causes frequent outbreaks. To investigate genetic changes in viral evolution, complete EV71 genome sequences were used to analyze the intra-genotypic evolution pattern in Taiwan, China, and the Netherlands.

Results

Genotype B5 was predominant in Taiwan’s 2008 outbreak and was re-emergent in 2012. EV71 strains from both outbreaks were phylogenetically segregated into two lineages containing fourteen non-synonymous substitutions predominantly in the non-structural protein coding region. In China, genotype C4 was first seen in 1998 and caused the latest large outbreak in 2008. Unlike shifting genotypes in Taiwan, genotype C4 persisted with progressive drift through time. A majority of non-synonymous mutations occurred in residues located in the non-structural coding region, showing annual increases. Interestingly, genotype B1/B2 in the Netherlands showed another stepwise evolution with dramatic EV71 activity increase in 1986. Phylogeny of the VP1 coding region in 1971–1986 exhibited similar lineage turnover with genotype C4 in China; however, phylogeny of the 3D-encoding region indicated separate lineage appearing after 1983, suggesting that the 3D-encoding region of genotype B2 was derived from an unidentified ancestor that contributed to intra-genotypic evolution in the Netherlands.

Conclusions

Unlike VP1 coding sequences long used for phylogenetic study of enteroviruses due to expected host immune escape, our study emphasizes a dominant role of non-synonymous mutations in non-structural protein regions that contribute to (re-)emergent genotypes in continuous stepwise evolution. Dozens of amino acid substitutions, especially in non-structural proteins, were identified via genetic changes driven through intra-genotypic evolution worldwide. These identified substitutions appeared to increase viral fitness in the population, affording valuable insights not only for viral evolution but also for prevention, control, and vaccine against EV71 infection.     

Intranasal Immunization of Baculovirus Displayed Hemagglutinin Confers Complete Protection against Mouse Adapted Highly Pathogenic H7N7 Reassortant Influenza Virus

Background

Avian influenza A H7N7 virus poses a pandemic threat to human health because of its ability for direct transmission from domestic poultry to humans and from human to human. The wide zoonotic potential of H7N7 combined with an antiviral immunity inhibition similar to pandemic 1918 H1N1 and 2009 H1N1 influenza viruses is disconcerting and increases the risk of a putative H7N7 pandemic in the future, underlining the urgent need for vaccine development against this virus.

Methodology/Principal Findings

In this study, we developed a recombinant vaccine by expressing the H7N7-HA protein on the surface of baculovirus (Bac-HA). The protective efficacy of the live Bac-HA vaccine construct was evaluated in a mouse model by challenging mice immunized intranasally (i.n.) or subcutaneously (s.c.) with high pathogenic mouse adapted H7N7 reassorted strain. Although s.c. injection of live Bac-HA induced higher specific IgG than i.n. immunization, the later resulted in an elevated neutralization titer. Interestingly, 100% protection from the lethal viral challenge was only observed for the mice immunized intranasally with live Bac-HA, whereas no protection was achieved in any other s.c. or i.n. immunized mice groups. In addition, we also observed higher mucosal IgA as well as increased IFN-γ and IL-4 responses in the splenocytes of the surviving mice coupled with a reduced viral titer and diminished histopathological signs in the lungs.

Conclusion

Our results indicated that protection from high pathogenic H7N7 (NL/219/03) virus requires both mucosal and systemic immune responses in mice. The balance between Th1 and Th2 cytokines is also required for the protection against the H7N7 pathogen. Intranasal administration of live Bac-HA induced all these immune responses and protected the mice from lethal viral challenge. Therefore, live Bac-HA is an effective vaccine candidate against H7N7 viral infections.     

Incidence rates of enterovirus 71 infections in young children during a nationwide epidemic in Taiwan, 2008-09

Objective

Enterovirus 71 (EV71) is causing life-threatening outbreaks in tropical Asia. In Taiwan and other tropical Asian countries, although nationwide EV71 epidemics occur cyclically, age-specific incidence rates of EV71 infections that are critical to estimate disease burden and design vaccine trials are not clear. A nationwide EV71 epidemic occurred in 2008–09 in Taiwan, which provided a unique opportunity to estimate age-specific incidence rates of EV71 infections.

Study Design

We prospectively recruited 749 healthy neonates and conducted follow-ups from June 2006 to December 2009. Sera were obtained from participants at 0, 6, 12, 24, and 36 months of age for measuring EV71 neutralizing antibody titers. If the participants developed suspected enterovirus illnesses, throat swabs were collected for virus isolation.

Results

We detected 28 EV71 infections including 20 symptomatic and 8 asymptomatic infections. Age-specific incidence rates of EV71 infection increased from 1.71 per 100 person-years at 0–6 months of age to 4.09, 5.74, and 4.97 per 100 person-years at 7–12, 13–24, and 25–36 months of age, respectively. Cumulative incidence rate was 15.15 per 100 persons by 36 months of age, respectively.

Conclusions

Risk of EV71 infections in Taiwan increased after 6 months of age during EV71 epidemics. The cumulative incidence rate was 15% by 36 months of age, and 29% of EV71 infections were asymptomatic in young children.     

Partially Neutralizing Potency against Emerging Genotype I Virus among Children Received Formalin-Inactivated Japanese Encephalitis Virus Vaccine

Background

Genotype I (GI) Japanese encephalitis virus (JEV) that replaced GIII virus has become the dominant circulating virus in Asia. Currently, all registered live and inactivated JEV vaccines are derived from genotype III viruses. In Taiwan, the compulsory JEV vaccination policy recommends that children receives four doses of formalin-inactivated Nakayama (GIII) JEV vaccine.

Methodology/Principal Findings

To evaluate the influence of genotype replacement on the post-vaccination viral neutralizing ability by GIII and GI viruses, the small panel of vaccinated-children serum specimens was assembled, and the reciprocal 50% plaque-reduction neutralizing antibody titers (PRNT₅₀) were measured against Nakayama vaccine strain, CJN GIII human brain isolate and TC2009-1 GI mosquito isolate. The seropositivity rate (PRNT₅₀≥1∶10) and geometric mean titers (GMT) against the TC2009-1 virus were the lowest among the three viruses. The protective threshold against the CJN and TC2009-1 viruses could only be achieved when the GMT against Nakayama virus was ≥1∶20 or ≥1∶80, respectively. Using undiluted vaccinees'' sera, the enhancement of JEV infection in K562 cells was observed in some low or non-neutralizing serum specimens.

Conclusions/Significance

Our preliminary study has shown that neutralizing antibodies, elicited by the mouse brain-derived and formalin-inactivated JEV Nakayama vaccine among a limited number of vaccinees, have reduced neutralizing capacity against circulating GI virus, but more detailed studies are needed to address the potential impact on the future vaccine policy.     

A Pandemic Influenza H1N1 Live Vaccine Based on Modified Vaccinia Ankara Is Highly Immunogenic and Protects Mice in Active and Passive Immunizations

Background

The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model.

Methodology/Principal Findings

For this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus - a safe poxviral live vector – resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-γ-secreting (IFN-γ) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus.

Conclusions/Significance

The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza.     

Display of VP1 on the surface of baculovirus and its immunogenicity against heterologous human enterovirus 71 strains in mice

Background

Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and has caused high mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no effective vaccine and antiviral agents available against EV71 infections. VP1 is one of the major immunogenic capsid protein of EV71 and plays a crucial role in viral infection. Antibodies against VP1 are important for virus neutralization.

Methodology/Principal Finding

In the present study, infectious EV71 viruses were generated from their synthetic complementary DNA using the human RNA polymerase I reverse genetics system. Secondly, the major immunogenic capsid protein (VP1) of EV71-Fuyang (subgenogroup C4) was displayed on the surface of recombinant baculovirus Bac-Pie1-gp64-VP1 as gp64 fusion protein under a novel White Spot Syndrome Virus (WSSV) immediate early ie1 promoter. Baculovirus expressed VP1 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that VP1 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired VP1 from the insect cell membrane via the budding process. After two immunizations in mice, Bac-Pie1-gp64-VP1 elicited neutralization antibody titer of 1∶64 against EV71 (subgenogroup C4) in an in vitro neutralization assay. Furthermore, the antisera showed high cross-neutralization activities against all 11 subgenogroup EV71 strains.

Conclusion

Our results illustrated that Bac-Pie1-gp64-VP1 retained native epitopes of VP1 and acted as an effective EV71 vaccine candidate which would enable rapid production without any biosafety concerns.