Intramuscular Inoculation of AS02-Adjuvanted Respiratory Syncytial Virus (RSV) F Subunit Vaccine Shows Better Efficiency and Safety Than Subcutaneous Inoculation in BALB/c Mice.

We previously explored a panel of adjuvants formulated with pre-fusion RSV-F protein and found that AS02 may be a promising candidate adjuvant for developing RSV-F subunit vaccines with improved immunogenicity and desired immune response type. In this study, we performed a head-to-head comparison of the effect of intramuscular injection to that of subcutaneous injection on the immune response and protective efficacy of recombinant RSV-F subunit vaccine with or without adjuvants (Alhydrogel, squalene-based emulsion adjuvants MF59, AS03, and AS02) in BALB/c mice. After inoculations, antigen-specific antibodies, neutralizing antibodies, antibody subtypes, cytokines, and the persistence of immune response were evaluated. Moreover, challenge tests were also performed to illustrate the possible effect of inoculation routes and adjuvant on virus clearance and histochemistry changes in the lungs of mice. The results indicated that intramuscular inoculation is a more effective and antigen dose-sparing route to enhance the immune response, although subcutaneous inoculation induced faster and stronger IgG antibodies after the initial immunization. Furthermore, adjuvant, but not immunization route, is a more critical factor to affect the humoral/cellular immune response and the immune bias. In addition, adjuvant inoculated via the intramuscular route is safer than that via the subcutaneous route, especially for AS02. This study highlights the importance of the adjuvant and immunization routes in the design and clinical transformation of adjuvanted vaccines. Further investigation is needed to illustrate the mechanism underlying the above difference in both efficiency and safety.

Respiratory Syncytial Virus F Subunit Vaccine With AS02 Adjuvant Elicits Balanced, Robust Humoral and Cellular Immunity in BALB/c Mice.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory illness, particularly in infants, the elderly, and immunocompromised adults. There is no licensed commercial vaccine against RSV. Importantly, formalin-inactivated RSV vaccines mediate enhanced respiratory disease. RSV fusion (F) protein with pre-fusion conformation is a promising candidate subunit vaccine. However, some problems remain to be solved, such as low immunogenicity and humoral immunity bias. Adjuvants can effectively enhance and adjust vaccine immune responses. In this study, we formulated pre-fusion RSV-F protein with the adjuvants, Alhydrogel, MF59, AS03, AS02, and glycol chitosan (GCS). We then conducted head-to-head comparisons of vaccine-induced immune responses in BALB/c mice. All adjuvanted vaccines enhanced antigen-specific and neutralizing antibody titers and viral clearance and gave an order of adjuvant activity: AS02 > AS03, MF59 > GCS, and Alhydrogel. Among them, AS02 elicited the highest antibody expression, which persisted until week 18. Moreover, AS02 significantly enhanced Th1 type immune response in immunized mice. Mice in the AS02 group also showed faster recovery from viral attacks in challenge tests. Further transcriptome analysis revealed that AS02 regulates immune balance by activating TLR-4 and promotes Th1-type immune responses. These results suggest that AS02 may be an excellent candidate adjuvant for RSV-F subunit vaccines. This study also provides valuable information regarding the effect of other adjuvants on immune responses of RSV-F subunit vaccines.

An HIV-1 vaccine based on bacterium-like particles elicits Env-specific mucosal immune responses.

Although many vaccines have been designed to induce effective mucosal immune responses against HIV-1, designing an effective HIV-1 vaccine remains a challenge. Bacterium-like particles (BLPs) are a new type of vector used to induce mucosal immune responses, and have already been used for some vaccines against respiratory tract viruses. In this study, we designed a mucosal vaccine against HIV-1 based on BLPs. The vaccine was used to immunize both mice and guinea pigs via intramuscular (i.m.) injection or intranasal (i.n.) drip. We found that gp120 trimers bound to BLPs delivered via i.n. drip successfully induced Env-specific secretory IgA (sIgA) at mucosal sites in mice. Furthermore, nasal washes from guinea pigs immunized via i.n. drip showed neutralizing activity against HIV-1 tier 1 pseudoviruses. Thus, gp120 trimers bound to BLPs may be an effective vaccine strategy against HIV-1.

Broad protective immune responses elicited by bacterium-like particle-based intranasal pneumococcal particle vaccine displaying PspA2 and PspA4 fragments.

Streptococcus pneumoniae is an infectious pathogen mainly infecting host bodies through the respiratory system. An effective pneumococcal vaccine would be targeted to the mucosa and provide not only protection against invasive infection but also against colonization in the respiratory system. In the present work, we applied bacterium-like particles (BLPs) as an adjuvant for the development of a PspA mucosal vaccine, in which the PspA protein was displayed on the surface of BLPs. Intranasal immunization with the PspA-BLP pneumococcal vaccine, comprised of PspA2 from pneumococcal family 1 and PspA4 from pneumococcal family 2, not only induced a high level of serum IgG antibodies but also a high level of mucosal SIgA antibodies. Analysis of binding of serum antibodies to intact bacteria showed a broad coverage of binding to pneumococcal strains expressing PspA from clade 1 to 5. Immunization with the PspA-BLP vaccine conferred protection against fatal intranasal challenge with both PspA family 1 and family 2 pneumococcal strains regardless of serotype. Therefore, the PspA-BLP pneumococcal vaccine was demonstrated to be a promising strategy for mucosal immunization to enhance both systemic and mucosal immune responses.

Expression and purification of pneumococcal surface protein a of clade 4 in Escherichia coli using hydroxylapatite and ion-exchange column chromatography.

Streptococcus pneumoniae is a major pathogen that causes life-threatening diseases, such as pneumonia, otitis media, bacteremia, and meningitis, worldwide and especially in young children and the elderly. Pneumococcal surface protein A (PspA) is a widely studied candidate protein vaccine that represents a promising replacement for current polysaccharide and polysaccharide-conjugate vaccines. In this study, we describe a simple method to produce PspA of clade 4 from an Escherichia coli expression system using hydroxylapatite and ion-exchange chromatography. Using this method, we successfully expressed soluble PspA4 in 10 L of autoinducing culture medium, with a wet-cell yield of 19 g/L and a final PspA4 concentration of 22.8 mg/L. Additionally, we improved PspA4 purity from 17% to 70% in a single step through the use of hydroxylapatite, resulting in acquisition of recombinant PspA4 (>95% purity) at a final yield of 43% from the starting cell-lysis solution. We subsequently verified the secondary structure molecular weight of recombinant PspA4 by circular dichroism and mass spectrometry, respectively. These results demonstrated a highly efficient method for mass producing PspA4 protein and that can also be applied for purification of PspA proteins from other clades.

The Chinese giant salamander exemplifies the hidden extinction of cryptic species.

Overexploitation, habitat destruction, human-driven climate change and disease spread are resulting in the extinction of innumerable species, with amphibians being hit harder than most other groups [1]. Few species of amphibians are widespread, and those that are often represent complexes of multiple cryptic species. This is especially true for range-restricted salamanders [2]. Here, we used the widespread and critically endangered Chinese giant salamander (Andrias davidianus) to show how genetically uninformed management efforts can negatively affect species conservation. We find that this salamander consists of at least five species-level lineages. However, the extensive recent translocation of individuals between farms, where the vast majority of extant salamanders now live, has resulted in genetic homogenization. Mitochondrial DNA (mtDNA) haplotypes from northern China now predominate in farms. Unfortunately, hybrid offspring are being released back into the wild under well-intentioned, but misguided, conservation management. Our findings emphasize the necessity of genetic assessments for seemingly well-known, widespread species in conservation initiatives. Species serve as the primary unit for protection and management in conservation actions [3], so determining the taxonomic status of threatened species is a major concern, especially for amphibians. The level of threat to amphibians may be underestimated, and existing conservation strategies may be inadvertently harmful if conducted without genetic assessment.

Imminent extinction in the wild of the world's largest amphibian.

Species with large geographic ranges are considered resilient to global decline [1]. However, human pressures on biodiversity affect increasingly large areas, in particular across Asia, where market forces drive overexploitation of species [2]. Range-wide threat assessments are often costly and thus extrapolated from non-representative local studies [3]. The Chinese giant salamander (Andrias davidianus), the world's largest amphibian, is thought to occur across much of China, but populations are harvested for farming as luxury food [4]. Between 2013 and 2016, we conducted field surveys and 2,872 interviews in possibly the largest wildlife survey conducted in China. This extensive effort revealed that populations of this once-widespread species are now critically depleted or extirpated across all surveyed areas of their range, and illegal poaching is widespread.

Comparison of Immunogenicity and Protection of Two Pneumococcal Protein Vaccines Based on PsaA and PspA.

Streptococcus pneumoniae is a major cause of invasive pneumococcal disease, septicemia, and meningitis that can result in high morbidity rates in children under 5 years old. The current polysaccharide-based vaccines can provide type-specific immunity, but a broad-spectrum vaccine would provide greater coverage. Therefore, developing pneumococcal-protein-based vaccines that can extend to more serum types is highly important. In this study, we vaccinated mice via the subcutaneous (s.c.) route with a systemic vaccine that is a mixture of fusion protein PsaA-PspA23 and a single protein, PspA4, with aluminum hydroxide as an adjuvant. As a comparison, mice were immunized intranasally with a mucosal vaccine that is a mixture of PspA2-PA-BLP (where PA is protein anchor and BLP is bacterium-like particle) and PspA4-PA-BLP, via the intranasal (i.n.) route. The two immunization processes were followed by challenge with Streptococcus pneumoniae bacteria from two different PspA families. Specific IgG titers in the serum and specific IgA titers in the mucosa were determined following immunizations. Bacterial loads and survival rates after challenge were compared. Both the systemic vaccine and the mucosal vaccine induced a significant increase of IgG against PspAs. Only the mucosal vaccine also induced specific IgA in the mucosa. The two vaccines provided protection, but each vaccine showed an advantage. The systemic vaccine induced higher levels of serum antibodies, whereas the mucosal vaccine limited the bacterial load in the lung and blood. Therefore, coimmunizations with the two types of vaccines may be implemented in the future.

A Novel PspA Protein Vaccine Intranasal Delivered by Bacterium-Like Particles Provides Broad Protection Against Pneumococcal Pneumonia in Mice.

BACKGROUND: Streptococcus pneumoniae is a major pathogen accounting for a large number of pneumococcal disease in worldwide. Due to the mucosal immune pathway induces both systemic and mucosal immune responses, the potential strategy to prevent pneumococcal disease may be to develop a mucosal vaccine. METHOD: In this study, we developed an intranasal pneumococcal protein vaccine based on a bacterium-like particle (BLP) delivery system. PspA is expressed and exposed on the surface of all pneumococcal strains, which confers the potential to induce immune responses to protect against pneumococcal infection. We fused one of the pneumococcal surface proteins (PspA, family2 clade4) with the protein anchor (PA) protein in order to display PspA on the surface of BLPs. RESULT: The current results showed that intranasal immunization with BLPs/PspA-PA efficiently induced both PspA-specific IgG in the serum and PspA-specific IgA in mucosal washes. And intranasal immunization of BLPs/PspA-PA could provide complete protection in a mouse challenge model with pneumococci of different two clades of both homologous and heterologous PspA families. DISCUSSION AND CONCLUSION: Thus, targeted delivery of multiple bacterial antigens via BLPs may prevent pneumococcal disease by inducing both systemic and mucosal immune responses.

Systemic and mucosal immune responses elicited by intranasal immunization with a pneumococcal bacterium-like particle-based vaccine displaying pneumolysin mutant Plym2.

Pneumolysin (Ply) is an important virulence factor in pneumococcal infection and a conserved cholesterol-binding cytotoxin expressed by all serotypes of Streptococcus pneumoniae. We previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two amino acids (C428G and W433F), which lost cytotoxicity but retained the ability to induce neutralizing antibodies. In the present work, we applied bacterium-like particles (BLPs) as a carrier and immunostimulant for the development of a Plym2 intranasal vaccine, in which the Plym2 protein was displayed on the surface of BLPs. Intranasal immunization of mice with BLP-Plym2 not only induced a high level of serum IgG antibodies but also a high level of mucosal SIgA antibodies in lung lavages. Antiserum induced by the BLP-Plym2 vaccine elicited high-titer neutralization activity which could inhibit the hemolysis of wild-type Ply. In conclusion, the BLP-Plym2 vaccine was demonstrated to be a promising strategy for intranasal immunization to enhance both systemic and mucosal immune responses.

Protective Immune Responses Elicited by Fusion Protein Containing PsaA and PspA Fragments.

Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Due to drawbacks of the current polysaccharide-based vaccine, the most promising way to generate an improved vaccine may be to utilize protection-eliciting pneumococcal proteins. Pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA) are two vaccine candidates which have been evaluated against S. pneumoniae infection in animal models or human clinical trials with encouraging results. In this study, the efficacy of the fusion protein PsaA-PspA, which includes PsaA part and PspA part, in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in an animal model. PspA part of PsaA-PspA fusion protein contains both family1 N-terminal region and family 2 N-terminal clade-defining region of PspA. Immunization with the PsaA-PspA fusion protein induced high levels of antibodies against both PsaA and PspA, which could bind to intact S. pneumoniae strains bearing different PspAs. Ex vivo stimulation of splenocytes from mice immunized with PsaA-PspA induced IL-17A secretion. Mice immunized with PsaA-PspA showed reduced S. pneumoniae levels in the blood and lungs compared with the PBS group after intranasal infection. Finally, mice immunized with PsaA-PspA fusion proteins were protected against fatal challenge with pneumococcal strains expressing different PspAs regardless of the challenge route. These results support the PsaA-PspA fusion protein as a promising vaccine strategy, as demonstrated by its ability to enhance the immune response and stimulate production of high titer antibodies against S. pneumoniae strains bearing heterologous PspAs, as well as confer protection against fatal challenge with PspA family 1 and family 2 strains.

Immunogenicity and protective efficacy of an EV71 virus-like particle vaccine against lethal challenge in newborn mice.

Enterovirus 71(EV71) has caused severe epidemics of hand, foot and mouth disease (HFMD) in the Asia Pacific in recent years, particularly in infants and pre-school children. It has become a serious public health threat, as currently there are no approved vaccines or antiviral drugs for EV71 infection. Many EV71 vaccines have been under development worldwide, however the main focus is inactivated EV71 vaccines. For example, the inactivated EV71 vaccine has recently finished phase III clinical trial in Mainland China. There have been very few studies on EV71 virus like particles (VLPs). In this study, the immunogenicity and protective potency of the EV71 VLPs produced in insect cells were evaluated in mice with different dosages. Our results showed that EV71 VLPs could elicit high titers of neutralizing antibodies (NTAbs) in a dose-dependent manner and NTAbs were sustained after the second injection with an average GMT (geometric mean titer) level from 19 to 2960 in immunized mice. Survival rates were 100%, 100%, 85%, and 40% after challenge with 15 LD50 (median lethal dose) of EV71 in these newborn mice, respectively. ED50 (50% effective dose) of VLPs was 0.20 µg/dose in newborn mice, while NTAb titer under this dosage was about 50. Passive protection was determined with 2 methods and demonstrated that the survival rates were positively correlated with NTAb titers, which at 24 and 54 induced 50% survival rates in experimental animals. The ED50 of VLP vaccines and the passive NTAb titers were also analyzed. The maternal NTAb titer was similar as the passive NTAb titer in the mouse model challenged with our lethal mouse EV71 strain. Hence, our work has provided preliminary data on the protection potency of VLPs as a vaccine candidate and would facilitate future VLP vaccine development.

Evaluation of monovalent and bivalent vaccines against lethal Enterovirus 71 and Coxsackievirus A16 infection in newborn mice.

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) have caused severe epidemics of hand, foot and mouth disease (HFMD) in the Asia Pacific in recent years, particularly in infants and young children. This disease has become a serious public health problem, as no vaccines or antiviral drugs have been approved for EV71 and CA16 infections. In this study, we compared four monovalent vaccines, including formalin-inactivated EV71 virus (iEV71), EV71 virus-like particles (VLPs) (vEV71), formalin-inactivated CVA16 virus (iCVA16) and CVA16 VLPs (vCVA16), along with two bivalent vaccines, including equivalent doses of formalin-inactivated EV71+CVA16 virus (iEV71+iCVA16) and EV71+CVA16 VLPs (vEV71+vCVA16). The IgG titers and neutralization antibodies titers demonstrated that there are no immune interference exists between the two immunogens of EV71 and CVA16. IgG subclass isotyping revealed that IgG1 and IgG2b were induced primarily in all vaccine groups. Furthermore, cross-neutralization antibodies were elicited in mouse sera against other sub-genotypes of EV71 and CVA16. In vivo challenge experiments showed that the immune sera from vaccinated animals could confer passive protection to newborn mice against lethal challenge with 14 LD50 of EV71 and 50 LD50 of CVA16. Our results indicated that bivalent vaccination is promising for HFMD vaccine development. With the advantage of having a better safety profile than inactivated virus vaccines, VLPs should be used to combine both EV71 and CVA16 antigens as a candidate vaccine for prevention of HFMD virus transmission.

Detoxified pneumolysin derivative Plym2 directly protects against pneumococcal infection via induction of inflammatory cytokines.

Streptococcus pneumoniae is a major cause of infectious disease and complications worldwide, such as pneumonia, otitis media, bacteremia and meningitis. New generation protein-based pneumococcal vaccines are recognized as alternative vaccine candidates. Pneumolysin (Ply) is a cholesterol-dependent cytolysin produced by all clinical isolates of S. pneumoniae. Our research group previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two animo acids (C428G and W433F). Exhibiting undetectable levels of cytotoxicity, Plym2 could still elicit high titer neutralizing antibodies against the native toxin. However, evaluation of the active immunoprotective effects of Plym2 by subcutaneous immunization and lethal challenge with S. pneumoniae in mice did not yield favorable results. In the present work, we confirmed the previous observations by using passive immunization and systemic challenge. Results of the passive immunization were consistent with those of active immunization. Further experiments were conducted to explain the inability of high titer neutralizing antibodies against Ply to protect mice from S. pneumoniae challenge. Pneumococcal Ply is known to be the major factor responsible for the induction of inflammation that benefits the host. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes at the early infection stage. We demonstrated that Plym2 could induce proinflammatory cytokines similarly to wild-type Ply. A systemic infection model was used to clarify that Plym2 lacking cytolytic activity could protect mice from intraperitoneal challenge directly, while antibodies to the mutant had no effect. Therefore, the protective function of Plym2 may be due to its induction of proinflammatory cytokines. When used in the systemic infection model, Plym2 antibodies may block the induction of proinflammatory cytokines by Ply. These findings demonstrate that a Ply-based vaccine would not be an effective primary vaccine component, but it may be beneficial as an adjuvant to stimulate cytokine production.

New occurrence of Sinovipera sichuanensis in Guizhou.

Three Asian green pit vipers were collected in August 2013 during a field trip in Fanjin Mt. National Conservation Area, Guizhou. These specimens were identified as Sinovipera sichuanensis, based on subsequent examination and comparison. This is a new record of the genus Sinovipera and S. sichuanensis in Guizhou, and the first time that male specimens have been collected in the field.

Isolation and structural elucidation of proline-containing cyclopentapeptides from an endolichenic Xylaria sp.

Two new cyclic pentapeptides (1 and 2) and the known blazein (3), ganodesterone (4), ergosterin (5), cerevisterol (6), 24-methylcholesta-4,6,8(14),22-tetraen-3-one (7), 5,8-epidioxyergosta-6,22-dien-3-ol (8), 16-α-d-mannopyranosyloxyisopimar-7-en-19-oic acid (9), and 16-hydroxy isopimar-7-en-19-oic acid (10) have been isolated from the crude extract of an endolichenic Xylaria sp. The structures of 1 and 2 were elucidated primarily by NMR and MS methods. The absolute configurations of 1 and 2 were assigned using Marfey's method on their acid hydrolysate. Compounds 1-10 were evaluated for activity against fungi and for synergistic antifungal activity. Compound 1 showed synergistic antifungal activity against Candida albicans SC5314 with 0.004 µg/mL ketoconazole.

[Bioavailability of cadmium in seawater to Paralichthys olivaceus].

With exposure experiment, this paper studied the accumulation and elimination of cadmium (Cd) in the viscera, muscle and gill of Paralichthys olivaceus, and the effects of TOC concentration in seawater on the Cd accumulation. The results showed that when the exposure concentration of Cd was 0.5 mg x L(-1), its accumulation in test tissues increased with time, and reached equilibrium on the 10th day. The accumulation was in order of viscera (147.73 mg x kg(-1) DW) > gill (15.55 mg x kg(-1) DW) > muscle (4.578 mg x kg(-1) DW), which meant that viscera was the main place for Cd accumulation. Determinations after moving the fish into clear seawater on the 13th day of exposure showed that due to depuration, the Cd accumulation in test tissues decreased with time. On the 15th day after moving, the Cd elimination was in order of viscera (80.66%) > gill (73.66%) > muscle (56.84%), suggesting that the Cd accumulation in Paralichthys olivaceus could be reduced to meet safe food standard. The TOC concentration in seawater had a significant effect on Cd accumulation, i.e., the accumulation of Cd decreased significantly with increasing TOC concentration, demonstrating that the TOC in seawater could decrease the bioavailability of cadmium.