Immunogenicity and protective efficacy of inactivated coxsackievirus B4 viral particles.

Coxsackievirus B4 (CVB4) is associated with a range of acute and chronic diseases such as hand, foot, and mouth disease, myocarditis, meningitis, pancreatitis, and type 1 diabetes, affecting millions of young children annually around the world. However, no vaccine is currently available for preventing CVB4 infection. Here, we report the development of inactivated viral particle vaccines for CVB4. Two types of inactivated CVB4 particles were prepared from CVB4-infected cell cultures as vaccine antigens, including F-particle (also called mature virion) consisting of VP1, VP3, VP2, and VP4 subunit proteins, and E-particle (also called empty capsid) which is made of VP1, VP3, and uncleaved VP0. Both the inactivated CVB4 F-particle and E-particle were able to potently elicit neutralizing antibodies in mice, despite slightly lower neutralizing antibody titres seen with the E-particle vaccine after the third immunization. Importantly, we demonstrated that passive transfer of either anti-F-particle or anti-E-particle sera could completely protect the recipient mice from lethal CVB4 challenge. Our study not only defines the immunogenicity and protective efficacy of inactivated CVB4 F-particle and E-particle but also reveals the central role of neutralizing antibodies in anti-CVB4 protective immunity, thus providing important information that may accelerate the development of inactivated CVB4 vaccines.

Synbiotics improve growth performance and nutrient digestibility, inhibit PEDV infection, and prevent intestinal barrier dysfunction by mediating innate antivirus immune response in weaned piglets.

This experiment was conducted to explore the effects of dietary synbiotics (SYB) supplementation on growth performance, immune function, and intestinal barrier function in piglets challenged with porcine epidemic diarrhea virus (PEDV). Forty crossbred (Duroc × Landrace × Yorkshire) weaned piglets (26 ±â€…1 d old) with a mean body weight (BW) of 6.62 ±â€…0.36 kg were randomly allotted to five groups: control (CON) I and CONII group, both fed basal diet; 0.1% SYB group, 0.2% SYB group, and 0.2% yeast culture (YC) group, fed basal diet supplemented with 0.1%, 0.2% SYB, and 0.2% YC, respectively. On day 22, all piglets were orally administrated with 40 mL PEDV (5.6 × 103 TCID50/mL) except piglets in CONI group, which were administrated with the same volume of sterile saline. The trial lasted for 26 d. Before PEDV challenge, dietary 0.1% SYB supplementation increased final BW, average daily gain (ADG), and decreased the ratio of feed to gain during 0 to 21 d (P < 0.05), as well as improved the apparent nutrient digestibility of dry matter (DM), organic matter (OM), crude protein, ether extract (EE), and gross energy (GE). At the same time, 0.2% YC also improved the apparent nutrient digestibility of DM, OM, EE, and GE (P < 0.05). PEDV challenge increased diarrhea rate and diarrhea indexes while decreased ADG (P < 0.05) from days 22 to 26, and induced systemic and intestinal mucosa innate immune and proinflammatory responses, destroyed intestinal barrier integrity. The decrease in average daily feed intake and ADG induced by PEDV challenge was suppressed by dietary SYB and YC supplementation, and 0.1% SYB had the best-alleviating effect. Dietary 0.1% SYB supplementation also increased serum interleukin (IL)-10, immunoglobulin M, complement component 4, and jejunal mucosal IL-4 levels, while decreased serum diamine oxidase activity compared with CONII group (P < 0.05). Furthermore, 0.1% SYB improved mRNA expressions of claudin-1, zonula occludens protein-1, mucin 2, interferon-γ, interferon regulatory factor-3, signal transducers and activators of transcription (P < 0.05), and protein expression of occludin, and downregulated mRNA expressions of toll-like receptor 3 and tumor necrosis factor-α (P < 0.05) in jejunal mucosa. Supplementing 0.2% SYB or 0.2% YC also had a positive effect on piglets, but the effect was not as good as 0.1% SYB. These results indicated that dietary 0.1% SYB supplementation improved growth performance under normal conditions, and alleviated the inflammatory response and the damage of intestinal barrier via improving innate immune function and decreasing PEDV genomic copies, showed optimal protective effects against PEDV infection.

Porcine epidemic diarrhea virus (PEDV) infection causes watery diarrhea, vomiting, anorexia, and high mortality in piglets, which leads to serious economic losses in many pig-producing countries. Vaccination is commonly used for the prevention of PEDV infection. However, current vaccines are ineffective in preventing infections because of genetic variants of PEDV. Therefore, developing new and efficient strategies to reduce porcine epidemic diarrhea outbreaks for piglets is desirable. Synbiotics (SYB) refer to the biological mixture of probiotics and prebiotics, which combines the advantages of both. At present, the application of probiotics or prebiotics has been widely reported in piglets feeds, which improves growth performance, immune function, microbiota community, intestinal structure, and resistance to bacterial infection. However, there was little report on whether SYB can protect piglets against PEDV infection. Therefore, this study was conducted to investigate the effects of SYB on growth performance, intestinal barrier function, and immune function in PEDV-infected weaned piglets. Results indicated that dietary SYB supplementation improved growth performance, decreased the inflammatory response, and alleviated the damage of intestinal barrier by improving innate antiviral immunity and reducing PEDV genomic copies, ultimately offering optimal protective effects against PEDV infection.

Recombinant proteins A29L, M1R, A35R, and B6R vaccination protects mice from mpox virus challenge.

Since May 2022, mutant strains of mpox (formerly monkeypox) virus (MPXV) have been rapidly spreading among individuals who have not traveled to endemic areas in multiple locations, including Europe and the United States. Both intracellular and extracellular forms of mpox virus have multiple outer membrane proteins that can stimulate immune response. Here, we investigated the immunogenicity of MPXV structural proteins such as A29L, M1R, A35R, and B6R as a combination vaccine, and the protective effect against the 2022 mpox mutant strain was also evaluated in BALB/c mice. After mixed 15 µg QS-21 adjuvant, all four virus structural proteins were administered subcutaneously to mice. Antibody titers in mouse sera rose sharply after the initial boost, along with an increased capacity of immune cells to produce IFN-γ alongside an elevated level of cellular immunity mediated by Th1 cells. The vaccine-induced neutralizing antibodies significantly inhibited the replication of MPXV in mice and reduced the pathological damage of organs. This study demonstrates the feasibility of a multiple recombinant vaccine for MPXV variant strains.

Single-dose rAAV5-based vaccine provides long-term protective immunity against SARS-CoV-2 and its variants.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus and its variants, has posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against SARS-CoV-2 variants. Therefore, novel vaccines to match mutated viral lineages by providing long-term protective immunity are urgently needed. We designed a recombinant adeno-associated virus 5 (rAAV5)-based vaccine (rAAV-COVID-19) by using the SARS-CoV-2 spike protein receptor binding domain (RBD-plus) sequence with both single-stranded (ssAAV5) and self-complementary (scAAV5) delivery vectors and found that it provides excellent protection from SARS-CoV-2 infection. A single-dose vaccination in mice induced a robust immune response; induced neutralizing antibody (NA) titers were maintained at a peak level of over 1:1024 more than a year post-injection and were accompanied by functional T-cell responses. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines produced high levels of serum NAs against the circulating SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta. A SARS-CoV-2 virus challenge showed that the ssAAV5-RBD-plus vaccine protected both young and old mice from SARS-CoV-2 infection in the upper and lower respiratory tracts. Whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genomes of vaccinated mice one year after vaccination, demonstrating vaccine safety. These results suggest that the rAAV5-based vaccine is safe and effective against SARS-CoV-2 and several variants as it provides long-term protective immunity. This novel vaccine has a significant potential for development into a human prophylactic vaccination to help end the global pandemic.

Transcutaneous immunization via dissolving microneedles protects mice from lethal influenza H7N9 virus challenge.

Avian influenza H7N9 virus has first emerged in 2013 and since then has spread in China in five seasonal waves. In humans, influenza H7N9 virus infection is associated with a high fatality rate; thus, an effective vaccine for this virus is needed. In the present study, we evaluated the usefulness of dissolving microneedles (MNs) loaded with influenza H7N9 vaccine in terms of the dissolution time, insertion capacity, insertion depth, and structural integrity of H7N9 virus in vitro. Our in vitro results showed MNs dissolved within 6 mins. The depth of skin penetration was 270 µm. After coating with a matrix material solution, the H7N9 proteins were agglomerated. We detected the H7N9 delivery time and humoral immune response in vivo. In a mouse model, the antigen retention time was longer for MNs than for intramuscular (IM) injection. The humoral response showed that similar to IM administration, MN administration increased the levels of functional and systematic antibodies and protection against the live influenza A/Anhui/01/2013 virus (Ah01/H7N9). The protection level was determined by the analysis of pathological sections of infected lungs. MN and IM administration yielded results superior to those in the control group. Taken together, these findings demonstrate that the use of dissolving MNs to deliver influenza H7N9 vaccines is a promising immunization approach.

In ovo nano-silver and nutrient supplementation improves immunity and resistance against Newcastle disease virus challenge in broiler chickens.

Silver nanoparticles (AgNPs) interact with the microbes and host immune system to protect against diseases. Fertile broiler eggs (n = 900) were allotted to six groups: un-injected control, sham (sterile water), AgNPs (50 µg), AgNPs+Amino acids (Methionine-10 mg + Arginine-25 mg), AgNPs+Vitamins (Vit B1-72µg + Vit B6-140µg), and AgNPs+Trace Elements (Zn-80 µg and Se-0.3 µg) and incubated for 18 days. On 18th embryonic day, 0.6 ml test solution was injected at the broad end of egg using 25 mm needle and transferred to hatcher. Post-hatch, half of the chicks from each group were vaccinated with Newcastle disease (ND) vaccine, and the other half were kept as unvaccinated unit and reared for 42 d with standard management practices. Hatchability, 1st and 42nd d body weight, feed intake, and feed conversion ratio were similar between treatment groups in both vaccinated and unvaccinated units. The relative weight of bursa Fabricius and thymus was similar, but spleen weight was higher (P ≤ 0.05) in AgNPs, AgNPs+Vits, and AgNPs+TEs chicks than control group. Cellular immune response (against mitogen phytohemagglutinin-P) was higher (P ≤ 0.05) in AgNPs+TEs chicks, whereas HA titer against sheep red blood cells antigen, serum IgG, IgM, and HI titer against ND vaccine was apparently higher in AgNPs+Vits group chicks than control. No clinical symptoms were observed in the vaccinated groups except for a few control birds 6 days postchallenge (PC). Three days PC, unvaccinated birds show depression, off feed, greenish diarrhea, and nasal discharge and the control group started dying. The highest cumulative infection (CI) was observed in sham (79.17%) and un-injected control (75%), but lowest in AgNPs+AAs birds (58.33%) on 3rd dpi. The CI reached 100% on 5th dpi in control groups and AgNPs, and 91.67% and 93.75% in AgNPs+TEs and AgNPs+AAs group, respectively. The AgNPs+TEs and AgNPs+AAs group birds lived for more than 90 h compared to 75 h in control groups and also had higher IL-6 and IL-2 gene expressions at 24 h PC. It was concluded that 50 µg/egg AgNPs with vitamins (B1 and B6) and trace elements (Zn and Se) improved performance, but AgNPs with trace elements and amino acids enhanced immune response and resistance against vND virus challenge in broilers.

Juvenile Wels Catfish (Silurus glanis) Display Age-Related Mortality to European Catfish Virus (ECV) under Experimental Conditions.

We have limited knowledge about the course of the European catfish virus (ECV) infection in different age groups of wels catfish (Silurus glanis). The results of this study demonstrate that an ECV strain isolated from the brown bullhead (Ameiurus nebulosus) in Hungary could cause devastating losses among juvenile wels catfish. Furthermore, the age-related mortality rate following ECV infection was investigated in three virus challenge experiments at two different virus dosages. Eight-week-old (ca. 3 g), ten-week-old (ca. 8 g), and sixteen-week-old (ca. 55 g) catfish were infected with ECV at 21°C. In the youngest age group, 96% (at a 106 TCID50/mL dosage) and 100% (at 105 TCID50/mL) mortality rates were observed, while these rates were reduced to 56% and 68% in the ten-week-old groups, respectively. The mortality was significantly higher in the virus-exposed groups than in the control ones. In the sixteen-week-old group, 23% mortality was detected at a 105 TCID50/mL concentration of ECV. Here, a significant difference was not found between the exposed and control groups. The performed experiments show that different age groups of wels catfish may have various susceptibility to ECV. These findings draw attention to the importance of the prevention of/protection against virus infections in juvenile (up to 3-month-old) wels catfish in aquaculture.

One or two dose regimen of the SARS-CoV-2 synthetic DNA vaccine INO-4800 protects against respiratory tract disease burden in nonhuman primate challenge model.

Safe and effective vaccines will provide essential medical countermeasures to tackle the COVID-19 pandemic. Here, we assessed the safety, immunogenicity and efficacy of the intradermal delivery of INO-4800, a synthetic DNA vaccine candidate encoding the SARS-CoV-2 spike protein in the rhesus macaque model. Single and 2 dose vaccination regimens were evaluated. Vaccination induced both binding and neutralizing antibodies, along with IFN-γ-producing T cells against SARS-CoV-2. Upon administration of a high viral dose (5 × 106 pfu) via the intranasal and intratracheal routes we observed significantly reduced virus load in the lung and throat, in the vaccinated animals compared to controls. 2 doses of INO-4800 was associated with more robust vaccine-induced immune responses and improved viral protection. Importantly, histopathological examination of lung tissue provided no indication of vaccine-enhanced disease following SARS-CoV-2 challenge in INO-4800 immunized animals. This vaccine candidate is currently under clinical evaluation as a 2 dose regimen.

Intranasal Nanoparticle Vaccination Elicits a Persistent, Polyfunctional CD4 T Cell Response in the Murine Lung Specific for a Highly Conserved Influenza Virus Antigen That Is Sufficient To Mediate Protection from Influenza Virus Challenge.

Lung-localized CD4 T cells play a critical role in the control of influenza virus infection and can provide broadly protective immunity. However, current influenza vaccination strategies primarily target influenza hemagglutinin (HA) and are administered peripherally to induce neutralizing antibodies. We have used an intranasal vaccination strategy targeting the highly conserved influenza nucleoprotein (NP) to elicit broadly protective lung-localized CD4 T cell responses. The vaccine platform consists of a self-assembling nanolipoprotein particle (NLP) linked to NP with an adjuvant. We have evaluated the functionality, in vivo localization, and persistence of the T cells elicited. Our study revealed that intranasal vaccination elicits a polyfunctional subset of lung-localized CD4 T cells that persist long term. A subset of these lung CD4 T cells localize to the airway, where they can act as early responders following encounter with cognate antigen. Polyfunctional CD4 T cells isolated from airway and lung tissue produce significantly more effector cytokines IFN-γ and TNF-α, as well as cytotoxic functionality. When adoptively transferred to naive recipients, CD4 T cells from NLP:NP-immunized lung were sufficient to mediate 100% survival from lethal challenge with H1N1 influenza virus. IMPORTANCE Exploiting new, more efficacious strategies to potentiate influenza virus-specific immune responses is important, particularly for at-risk populations. We have demonstrated the promise of direct intranasal protein vaccination to establish long-lived immunity in the lung with CD4 T cells that possess features and positioning in the lung that are associated with both immediate and long-term immunity, as well as demonstrating direct protective potential.

Induction of protective immune responses against a lethal Zika virus challenge post-vaccination with a dual serotype of recombinant vesicular stomatitis virus carrying the genetically modified Zika virus E protein gene.

The development of a vaccine to prevent Zika virus (ZIKV) infection has been one of the priorities in infectious disease research in recent years. There have been numerous attempts to develop an effective vaccine against ZIKV. It is imperative to choose the safest and the most effective ZIKV vaccine from all candidate vaccines to control this infection globally. We have employed a dual serotype of prime-boost recombinant vesicular stomatitis virus (VSV) vaccine strategy, to develop a ZIKV vaccine candidate, using a type 1 IFN-receptor knock-out (Ifnar-/-) mouse model for challenge studies. Prime vaccination with an attenuated recombinant VSV Indiana serotype (rVSVInd) carrying a genetically modified ZIKV envelope (E) protein gene followed by boost vaccination with attenuated recombinant VSV New Jersey serotype (rVSVNJ) carrying the same E gene induced robust adaptive immune responses. In particular, rVSV carrying the ZIKV E gene with the honeybee melittin signal peptide (msp) at the N terminus and VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the C terminus of the E gene induced strong protective immune responses. This vaccine regimen induced highly potent neutralizing antibodies and T cell responses in the absence of an adjuvant and protected Ifnar-/- mice from a lethal dose of the ZIKV challenge.

Bat influenza vectored NS1-truncated live vaccine protects pigs against heterologous virus challenge.

Swine influenza is an important disease for the swine industry. Currently used whole inactivated virus (WIV) vaccines can induce vaccine-associated enhanced respiratory disease (VAERD) in pigs when the vaccine strains mismatch with the infected viruses. Live attenuated influenza virus vaccine (LAIV) is effective to protect pigs against homologous and heterologous swine influenza virus infections without inducing VAERD but has safety concerns due to potential reassortment with circulating viruses. Herein, we used a chimeric bat influenza Bat09:mH3mN2 virus, which contains both surface HA and NA gene open reading frames of the A/swine/Texas/4199-2/1998 (H3N2) and six internal genes from the novel bat H17N10 virus, to develop modified live-attenuated viruses (MLVs) as vaccine candidates which cannot reassort with canonical influenza A viruses by co-infection. Two attenuated MLV vaccine candidates including the virus that expresses a truncated NS1 (Bat09:mH3mN2-NS1-128, MLV1) or expresses both a truncated NS1 and the swine IL-18 (Bat09:mH3mN2-NS1-128-IL-18, MLV2) were generated and evaluated in pigs against a heterologous H3N2 virus using the WIV vaccine as a control. Compared to the WIV vaccine, both MLV vaccines were able to reduce lesions and virus replication in lungs and limit nasal virus shedding without VAERD, also induced significantly higher levels of mucosal IgA response in lungs and significantly increased numbers of antigen-specific IFN-γ secreting cells against the challenge virus. However, no significant difference was observed in efficacy between the MLV1 and MLV2. These results indicate that bat influenza vectored MLV vaccines can be used as a safe live vaccine to prevent swine influenza.

Monitoring the performance of foot-and-mouth disease vaccines prepared against local strains in the face of antigenic evolution in the field.

National programs for foot-and-mouth disease (FMD) eradication includes the use of vaccination; Turkey which is endemic to FMD virus (FMDV) (except for the Thrace region) and there is a risk of incursion of exotic strains from eastern borders. In 2015, a devastating outbreak was caused by the A/ASIA/G-VII (G-VII) lineage, which led to the inclusion of a new vaccine strain (A/TUR/15) derived from this lineage in 3 months. Although most of the cattle population in Turkey was then immunized with A/TUR/15 (vaccine coverage: 92.8%), the G-VII lineage continued to cause outbreaks in the field despite the evidence of protection observed with A/TUR/15 in in vivo and in vitro tests. When G-VII field strains were examined, changes in their genomes were detected. As the lineage appeared to be evolving, an unconventional vaccination strategy was adapted which changed the vaccine strain with new variants of G-VII according to antigenic evolution. To assess the suitability of candidate vaccine strains derived from the variants of the G-VII lineage, three viral candidates were assessed (A/TUR/15, A/TUR/16 and A/TUR/17) by in vitro virus neutralization tests for r1 vaccine matching and in vivo heterologous challenge tests. Although all three vaccine strains were antigenically well matched with each other and other G-VII field viruses, due to continues outbreaks the vaccine strain was changed three times in 20 months from A/TUR/15 (Dec 2015) to A/TUR/16 (Dec 2016) and then to A/TUR/17 (Aug 2017). With this strategy serotype A has not been observed in the field since January 2018. This study highlights the importance of adapting the vaccine strains according to antigenic evolution as this could be a valuable combat strategy in endemic countries, rather than using well-known vaccine strain and relying only on the relationship coefficient (r1 ) value.

Inhibitory effects of recombinant porcine interferon-α on porcine transmissible gastroenteritis virus infections in TGEV-seronegative piglets.

Our previous research obtained purified recombinant porcine interferon-α (rPoIFN-α) containing thioredoxin (Trx) fusion tag in E. coli Rosetta (DE3). Here, we evaluate the efficacy of this rPoIFN-α to prevent piglets from the infection of the transmissible gastroenteritis virus (TGEV) attack. In this experiment, twenty-five TGEV-seronegative piglets were randomly divided into five groups. Group 1 was positive control and only challenged with TGEV; Pigs in groups 2-4 were pretreated with 2 × 10(7)IU/pig, 2 × 10(6)IU/pig, and 2 × 10(5)IU/pig rPoIFN-α before TGEV challenge. The fifth group is a negative control group. The animals of this group are pretreated only with Trx protein-containing PBS solution without TGEV challenge. After 48 h of rPoIFN-α pretreatment, the pigs in groups 1-4 were challenged by TGEV, and the pigs in group 5 were administered with PBS. The surveillance results show that Pigs pre-treated with 2 × 10 (7) IU/pig rPoIFN-α are fully aligned with the violent TGEV attack. Pigs pretreated with 2 × 10 (6) IU/pig rPoIFN-α are partially aligned with the violent TGEV attack. Though piglets pretreated with 2 × 10(6) IU/pig or 2 × 10(5)IU/pig rPoIFN-α cannot be adapted to the challenge of TGEV. However, the use of this dose of rPoIFN-α could put off the clinical signs of pigs than the positive control group of the above. These results indicate that rPoIFN-α can protect pigs from the infection of potential TGEV or delay the appearance of clinical symptoms, and its effect is dose-dependent.

Mourilyan virus pathogenicity in kuruma shrimp (Penaeus japonicus).

The bunyavirus Mourilyan virus (MoV) occurs commonly in Black tiger (Penaeus monodon) and kuruma shrimp (Penaeus japonicus) farmed in eastern Australia. There is circumstantial evidence of MoV causing mortalities among P. japonicus moved from farm ponds to tanks for rearing as broodstock. To directly assess its pathogenic potential, independent cohorts of pond- (n = 24) or tank-reared juvenile (n = 21) P. japonicus were challenged intramuscularly with a cephalothorax tissue homogenate of P. monodon containing high loads of MoV (1.48 ± 0.28 × 108 MoV RNA copies/µg total RNA). In each trial, mortalities accumulated gradually among the saline-injected controls. Mortality onset occurred 12-14 days earlier in the pond-reared shrimp, possibly due to them possessing low-level pre-existing MoV infections. Despite the time to onset of mortality differing, Kaplan-Meier survival analyses confirmed mortality rates to be significantly higher in both the pond- (p = .017) and tank-reared shrimp (p = .031) challenged with MoV. RT-qPCR data on shrimp sampled progressively over each trial showed high loads of MoV to establish following challenge and discounted GAV and other endemic viruses from contributing to mortality. Together, the data show that acute MoV infection can adversely compromise the survival of juvenile P. japonicus.

Characterization of humoral immune responses and degree of protection induced by influenza vaccine in cotton rats: Effects of low vaccine dose and single vs booster vaccination.

INTRODUCTION: Cotton rats are a suitable model for the study of influenza disease symptoms and responses to influenza vaccination. We have previously shown that two immunizations with 15 µg whole inactivated virus (WIV) influenza vaccine could completely protect animals from infection with the H1N1pdm09 virus. METHODS: To further explore the cotton rat model, we here investigated the protective potential of a single intramuscular immunization and of prime/boost intramuscular immunizations with a low amount of antigen. RESULTS: A single intramuscular immunization with doses more than or equal to 0.5 µg WIV reliably evoked antibody responses and doses more than or equal to 1 µg protected the animals from virus replication in the lungs and from severe weight loss. However, clinical symptoms like an increased respiration rate were still apparent. Administration of a booster dose significantly increased the humoral immune responses but did not or only moderately improved protection from clinical symptoms. CONCLUSION: Our data suggest that complete and partial protection by influenza vaccines can be mimicked in cotton rats by using specific vaccination regimens.

How virus size and attachment parameters affect the temperature sensitivity of virus binding to host cells: Predictions of a thermodynamic model for arboviruses and HIV.

Virus binding to host cells involves specific interactions between viral (glyco)proteins (GP) and host cell surface receptors (Cr) (protein or sialic acid (SA)). The magnitude of the enthalpy of association changes with temperature according to the change in heat capacity (ΔCp) on GP/Cr binding, being little affected for avian influenza virus (AIV) haemagglutinin (HA) binding to SA (ΔCp = 0 kJ/mol/K) but greatly affected for HIV gp120 binding to CD4 receptor (ΔCp = -5.0 kJ/mol/K). A thermodynamic model developed here predicts that values of ΔCp from 0 to ~-2.0 kJ/mol/K have relatively little impact on the temperature sensitivity of the number of mosquito midgut cells with bound arbovirus, while intermediate values of ΔCp of ~-3.0 kJ/mol/K give a peak binding at a temperature of ~20 °C as observed experimentally for Western equine encephalitis virus. More negative values of ΔCp greatly decrease arbovirus binding at temperatures below ~20 °C. Thus to promote transmission at low temperatures, arboviruses may benefit from ΔCp ~ 0 kJ/mol/K as for HA/SA and it is interesting that bluetongue virus binds to SA in midge midguts. Large negative values of ΔCp as for HIV gp120:CD4 diminish binding at 37 °C. Of greater importance, however, is the decrease in entropy of the whole virus (ΔSa_immob) on its immobilisation on the host cell surface. ΔSa_immob presents a repulsive force which the enthalpy-driven GP/Cr interactions weakened at higher temperatures struggle to overcome. ΔSa_immob is more negative (less favourable) for larger diameter viruses which therefore show diminished binding at higher temperatures than smaller viruses. It is proposed that small size phenotype through a less negative ΔSa_immob is selected for viruses infecting warmer hosts thus explaining the observation that virion volume decreases with increasing host temperature from 0 °C to 40 °C in the case of dsDNA viruses. Compared to arboviruses which also infect warm-blooded vertebrates, HIV is large at 134 nm diameter and thus would have a large negative ΔSa_immob which would diminish its binding at human body temperature. It is proposed that prior non-specific binding of HIV through attachment factors takes much of the entropy loss for ΔSa_immob so enhancing subsequent specific gp120:CD4 binding at 37 °C. This is consistent with the observation that HIV attachment factors are not essential but augment infection. Antiviral therapies should focus on increasing virion size, for example through binding of zinc oxide nanoparticles to herpes simplex virus, hence making ΔSa_immob more negative, and thus reducing binding affinity at 37 °C.

Broad Protection of Pigs against Heterologous PRRSV Strains by a GP5-Mosaic DNA Vaccine Prime/GP5-Mosaic rVaccinia (VACV) Vaccine Boost.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) viruses are a major cause of disease and economic loss in pigs worldwide. High genetic diversity among PRRSV strains is problematic for successful disease control by vaccination. Mosaic DNA and vaccinia (VACV) vaccines were developed in order to improve protection against heterologous PRRSV strains. METHODS: Piglets were primed and boosted with GP5-Mosaic DNA vaccine and recombinant GP5-Mosaic VACV (rGP5-Mosaic VACV), respectively. Pigs vaccinated with rGP5-WT (VR2332) DNA and rGP5-WT VACV, or empty vector DNA and empty VACV respectively, served as controls. Virus challenge was given to separate groups of vaccinated pigs with VR2332 or MN184C. Necropsies were performed 14 days after challenge. RESULTS: Vaccination with the GP5-Mosaic-based vaccines resulted in cellular reactivity and higher levels of neutralizing antibodies to both VR2332 and MN184C PRRSV strains. In contrast, vaccination of animals with the GP5-WT vaccines induced responses only to VR2332. Furthermore, vaccination with the GP5-Mosaic based vaccines resulted in protection against challenge with two heterologous virus strains, as demonstrated by the significantly lower viral loads in serum, tissues, porcine alveolar macrophages (PAMs), and bronchoalveolar lavage (BAL) fluids, and less severe lung lesions after challenge with either MN184C or VR2332, which have only 85% identity. In contrast, significant protection by the GP5-WT based vaccines was only achieved against the VR2332 strain. Conclusions: GP5-Mosaic vaccines, using a DNA-prime/VACV boost regimen, conferred protection in pigs against heterologous viruses.

Efficient Delivery of Dengue Virus Subunit Vaccines to the Skin by Microprojection Arrays.

Dengue virus is the most important arbovirus impacting global human health, with an estimated 390 million infections annually, and over half the world's population at risk of infection. While significant efforts have been made to develop effective vaccines to mitigate this threat, the task has proven extremely challenging, with new approaches continually being sought. The majority of protective, neutralizing antibodies induced during infection are targeted by the envelope (E) protein, making it an ideal candidate for a subunit vaccine approach. Using truncated, recombinant, secreted E proteins (sE) of all 4 dengue virus serotypes, we have assessed their immunogenicity and protective efficacy in mice, with or without Quil-A as an adjuvant, and delivered via micropatch array (MPA) to the skin in comparison with more traditional routes of immunization. The micropatch contains an ultra-high density array (21,000/cm2) of 110 µm microprojections. Mice received 3 doses of 1 µg (nanopatch, intradermal, subcutaneous, or intra muscular injection) or 10 µg (intradermal, subcutaneous, or intra muscular injection) of tetravalent sE spaced 4 weeks apart. When adjuvanted with Quil-A, tetravalent sE vaccination delivered via MPA resulted in earlier induction of virus-neutralizing IgG antibodies for all four serotypes when compared with all of the other vaccination routes. Using the infectious dengue virus AG129 mouse infectious dengue model, these neutralizing antibodies protected all mice from lethal dengue virus type 2 D220 challenge, with protected animals showing no signs of disease or circulating virus. If these results can be translated to humans, MPA-delivered sE represents a promising approach to dengue virus vaccination.

Towards a thermodynamic mechanistic model for the effect of temperature on arthropod vector competence for transmission of arboviruses.

Arboviruses such as West Nile virus (WNV), bluetongue virus (BTV), dengue virus (DENV) and chikungunya virus (CHIKV) infect their arthropod vectors over a range of average temperatures depending on the ambient temperature. How the transmission efficiency of an arbovirus (i.e. vector competence) varies with temperature influences not only the short term risk of arbovirus outbreaks in humans and livestock but also the long term impact of climate change on the geographical range of the virus. The strength of the interaction between viral surface (glyco)protein (GP) and the host cell receptor (Cr) on binding of virus to host cell is defined by the thermodynamic dissociation constant Kd_receptor which is assumed to equal 10-3 M (at 37 °C) for binding of a sialic acid (SA) on the arthropod midgut epithelial cell surface to a SA-binding site on the surface of BTV, for example. Here virus binding affinity is modelled with increasing number of GP/Cr contacts at temperatures from 10 °C to 35 °C taking into account the change in entropy on immobilization of the whole virus on binding (ΔSa_immob). Based on published data, three thermodynamic GP/Cr binding scenarios, namely enthalpy-driven, entropy-assisted and entropy-driven, are shown to affect the temperature sensitivity of virus binding in different ways. Thus for enthalpy-driven GP/Cr binding, viruses bind host cells much more strongly at 10 °C than 35 °C. A mechanistic model is developed for the number of arthropod midgut cells with bound virus and by building in a kinetic component for the rate of arbovirus replication and subsequent spread to the arthropod salivary glands, a model for the effect of temperature on vector competence is developed. The model separates the opposing effects of temperature on midgut cell binding affinity from the kinetic component of virogenesis. It successfully accommodates both increases in vector competence with temperature as for DENV and WNV in mosquitoes and decreases as for the CHIKV 2010-1909 strain in various populations of Aedes albopictus mosquitoes. Enhanced cell binding at lower temperatures through enthalpy-driven GP/Cr binding compensates for the lower replication rate to some degree such that some transmission can still occur at lower temperatures. In contrast, the strength of entropy-driven GP/Cr binding diminishes at low temperatures although there is no minimum temperature threshold for transmission efficiency. The magnitude of ΔSa_immob is an important data gap. It is concluded that thermodynamic and kinetic data obtained at the molecular level will prove important in modelling vector competence with temperature.

Oral vaccination through voluntary consumption of the convict grouper Epinephelus septemfasciatus with yeast producing the capsid protein of red-spotted grouper nervous necrosis virus.

Nervous necrosis viruses (NNV) cause serious economic losses in marine fish cultivation. The red-spotted grouper NNV (RGNNV) is the most common species of NNV worldwide. There have been many efforts to develop prophylactic NNV vaccines, and various types of vaccine candidate have been suggested. However, most were designed as injectable vaccines, which are not suitable for large-scale vaccination and cause too much stress to the fish. Oral vaccination through voluntary feeding is an ideal way to provide protective immunity to fish. In the present study, recombinant Saccharomyces cerevisiae producing RGNNV capsid protein was used as oral vaccine. The recombinant yeast was prepared in freeze-dried form after disruption. Convict groupers were divided into three groups, control, and oral and parenteral vaccination groups, each consisting of 700 fishes. The control group received no treatment, the parenteral group received one intraperitoneal injection of RGNNV virus-like particles, and the oral vaccination group consumed feed containing the lysed recombinant yeast; voluntary intake was allowed four times at one-week intervals. Both vaccination groups produced serum RGNNV neutralizing antibody titers of >103 (log 2, 9.96), sustained for at least 95days post-immunization. In addition, in response to challenge with RGNNV both groups suffered significantly reduced mortality and had reduced brain RGNNV titers. These results indicate that recombinant yeast-based oral fish vaccines have great potential for large-scale vaccination.