Cross-protective efficacy of inactivated whole influenza vaccines against Korean Y280 and Y439 lineage H9N2 viruses in mice

Since June 2020, the Y280 lineage H9N2 virus, which is distinct from the previously endemic Y439 lineage, has been circulating in poultry in Korea. In this study, we developed two whole inactivated vaccines, rgHS314 and vac564, against the Y280 and Y439 lineages, respectively, and evaluated their immunogenicity and protective efficacy against homologous or heterologous viral challenge in mice. Serum neutralizing antibody titers in the rgHS314-vaccinated group were higher (68 ± 8.4 10log₂) than in the vac564-vaccinated group (18 ± 8.4 10log₂). In homologous challenge, rgHS314 conferred 100% protection, with no severe clinical signs, no body weight loss, and no viral replication in any tissues tested except the nasal turbinate. Viral replication in the lungs at 1, 3, 5, and 7 days post-infection (dpi) was significantly lower than in the sham group (p < 0.01). By contrast, all mice in the sham group were dead by 8 dpi with severe clinical signs and weight loss. Likewise, vac564 conferred 100% protection with no weight loss and with significantly lower viral replication in the lung than in the sham group at 3 dpi (p < 0.01). However, both vaccines showed partial protection in heterologous challenge. Our results suggest that both the rgHS314 and vac564 vaccines could be candidate vaccines for further evaluation in humans.     

Carboxymethylated and acetylated xerogel derivatives of Plectranthus esculentus starch protect Newcastle disease vaccines against cold chain failure

Developing vaccine stabilizers from local natural sources is desirable especially if the stabilizer would enhance the ability of the antigen to withstand frequent failures in cold chains. The study was undertaken to formulate immunogenic live Newcastle Disease (ND) LaSota vaccines stabilized with modified native starches for use at cold and ambient temperatures and to assess the immunogenicity of the starch stabilized vaccines in vaccinated chickens. Native starch extracted from the tubers of Plectranthus esculentus (Family, Lamiaceae) was modified by carboxymethylation and acetylation/xerogel formation and used as vaccine stabilizers of ND LaSota virus with/without buffers/bulking excipients. Cold Chain Failure (CCF) was simulated by storing the vaccines at 5 ± 2 °C for one month then at 37 ± 1 °C for 96 h. The stability of the samples were evaluated in comparison with peptone stabilized ND vaccines using pH, residual moisture, XRD, reconstitution time, mean embryo infective dose (EID50) and haemagglutination (HA) tests. Haemagglutination inhibition was used to evaluate the efficacy of the vaccines in conferring positive serum antibody titers (≥2³ log₂) in vaccine-naïve 2-week old broilers that were orally administered a single dose of the vaccines kept at 37 ± 1 °C for 96 h and bled weekly over four weeks. Temperature, pH, moisture content and amorphousness impacted vaccine stability. Peptone stabilized vaccines were significantly less stable and most affected by temperature changes with 1.2log₁₀EID50 loss while buffered/bulked trehalose, carboxymethylated and acetylated/xerogelized starch stabilized vaccines were most stable (0.2–0.5log₁₀EID50 loss in titer) after 96 h in CCF. Buffered trehalose stabilized vaccine (TVB) had lower HA titres than peptone and starch stabilized vaccines containing D-mannitol and Na₂HPO₄. Antibody titres of vaccinated broilers were between 3.3 ± 1.398 and 8.35 ± 2.678. All the vaccines were immunogenic (HI ≥ 2³) and developed HI titres (≥2⁴) considered to be protective. Carboxymethylated and acetylated/xerogel derivatives of P. esculentus starch have a great potential as vaccine stabilizers especially in areas prone to CCF.     

Protective efficacy of vaccines of the Korea national antigen bank against the homologous H5Nx clade 2.3.2.1 and clade 2.3.4.4 highly pathogenic avian influenza viruses

The occurrence of severe outbreaks of highly pathogenic avian influenza in Korea led to establishment of a national antigen bank for emergency preparedness. Here, we developed five vaccines for this bank (clade 2.3.2.1C, clade 2.3.4.4A, B, C, and D) by reverse genetics, inactivated them with formalin, and evaluated the protective efficacy and potency of serial dilutions against lethal homologous challenge in specific-pathogen-free chickens. After vaccination with one dose, each vaccine resulted in 100% survival, with no clinical symptoms, or lack of detectable virus shedding, and high levels of pre-challenge protective immunity (8.4–10.2 log₂). After vaccination with one-tenth of the full dose, protection was similar to that with the full dose. After vaccination with one-hundredth of the initial dose, survival was 20–80%, and all vaccines showed virus shedding. Four vaccines (excluding clade 2.3.2.1C) had satisfactory potency. In antibody-persistence tests, all vaccines maintained long-lasting protective immunity. Our results suggest that inactivated reverse-genetics vaccines genetically matched to outbreak viruses provide adequate protection after a single vaccination.     

Immunogenicity and protective efficacy of virus-like particles and recombinant fiber proteins in broiler-breeder vaccination against fowl adenovirus (FAdV)-8b

Inclusion body hepatitis (IBH) is an economically important diseases in broiler chicken industry. Several serotypes of fowl adenovirus (FAdV) can cause IBH, among them, serotype FAdV-8b is associated with the majority of the IBH cases in Canada. Here, we evaluated FAdV-8b virus-like particles (VLPs) and recombinant FAdV-8b fiber proteins (expressed in E. coli) as potential broiler-breeder vaccines against IBH. For assessing the immunogenicity of vaccines, we investigated both humoral and cellular immunity. The humoral immune response was evaluated by determining total IgY and virus-neutralizing antibody in serum at 14, 28, 35 and 60 days post-immunization (dpi). We examined cellular immunity using flow cytometry by determining CD4:CD8 ratio change in peripheral blood after the booster vaccination. The protective effect of vaccines was tested by challenging 14 day-old progeny (n = 30/group) carrying maternal antibodies (MtAb) by challenging with virulent FAdV-8b virus (1 × 10⁷ TCID₅₀, FAdV-8b-SK). Although total IgY levels were comparable in all groups, the neutralizing antibody response in broiler-breeders at 35 and 60 dpi was significantly (p < 0.05) higher those vaccinated with FAdV-8b VLPs followed by FAdV-8b fiber compared to fiber-knob. Moreover, vaccines comprised of FAdV-8b VLPs and FAdV-8b fiber rather than FAdV-8b fiber-knob efficiently elicited the cell-mediated immune response as evidenced by a statistically significant (p < 0.05) CD8⁺ T-cell proliferative response in broiler-breeders four days after the booster vaccination. Unlike FAdV-8b fiber-knob, FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders were able to transfer a substantial amount (28.4 ± 9%) of MtAb to their progeny. Challenge revealed that MtAb provided 100% and 82.7% protection in progeny hatched from FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders, respectively. Collectively, our data suggest that FAdV-8b subunit vaccine-induced MtAb efficiently protected progeny against clinical IBH and broiler-breeder vaccination with subunit vaccines is a potential approach to protect against IBH.     

Immunogenicity of pulsatile-release PLGA microspheres for single-injection vaccination

The World Health Organization's Expanded Programme on Immunization has led to a dramatic rise in worldwide vaccination rates over the past 40 years, yet 19.4 million infants remain underimmunized each year. Many of these infants have received at least one vaccine dose but may remain unprotected because they did not receive subsequent booster doses due to logistical challenges. This study aimed to develop injectable controlled release microparticles with kinetics that mimic common vaccine dosing regimens consisting of large antigen doses administered periodically over the course of months in order to eliminate the need for boosters. Sixteen poly(lactic-co-glycolic acid) (PLGA) microsphere formulations containing bovine serum albumin (BSA) as a model vaccine antigen were screened in vitro to determine their respective release kinetics. Three formulations that exhibited desirable pulsatile release profiles were then selected for studying immunogenicity in mice. Two low-dose microsphere formulations induced peak anti-BSA IgG antibody titers of 13.9 ± 1.3 and 13.7 ± 2.2 log₂ compared to 15.5 ± 1.5 log₂ for a series of three bolus injections delivered at 0, 4, and 8 weeks with an equivalent cumulative dose. Similarly, high-dose formulations induced peak antibody titers that were 16.1 ± 2.1 log₂ compared to 17.7 ± 2.2 log₂ for controls. All three microparticle formulations studied in vivo induced peak antibody titers that were statistically similar to bolus controls. These results suggest that pulsatile antigen release from polymeric microparticles is a promising approach for single-injection vaccination, which could potentially reduce the logistical burden associated with immunization in the developing world.     

Sex difference in immune response to vaccination: A participant-level meta-analysis of randomized trials of IMVAMUNE® smallpox vaccine

IntroductionPrevious research shows immune response to vaccination differs by sex but this has not been explored for IMVAMUNE^®, a replication-deficient smallpox vaccine developed in response to the potential for bioterrorism using smallpox.MethodsWe conducted a participant-level meta-analysis (N = 275, 136 men, 139 women) of 3 randomized trials of IMVAMUNE conducted at 13 centers in the US through a federally-funded extramural research program. Studies were eligible for inclusion if they tested the standard dose (1 × 10⁸ TCID₅₀/mL on Days 0 and 28) of liquid formulation IMVAMUNE, were completed at the time of our search, and enrolled healthy vaccinia-naïve participants. Models of the peak log₂ ELISA and PRNT titers post-second vaccination were constructed for each study with sex as a covariate. Results from these models were combined into random effects meta-analyses of the sex difference in response to IMVAMUNE. We then compared this approach with fixed effects models using the combined participant level data.ResultsIn each study the mean peak log₂ ELISA titer was higher in men than women but no single study demonstrated a statistically significant difference. Combination of the adjusted study-specific estimates into the random effects model showed a higher mean peak log₂-titer in men compared with women (absolute difference [men–women]: 0.32, 95% CI: 0.02–0.60). Fixed effects models controlling for study showed a similar result (log₂ ELISA titer, men–women: 0.34, 95% CI: 0.04–0.63). This equates to a geometric mean peak titer that is approximately 27% higher in men than women (95% CI: 3–55%). Peak log₂ PRNT titers were also higher (although not significantly) in men (men–women: 0.14, 95% CI: −0.30 to 0.58).ConclusionOur results show statistically significant differences in response to IMVAMUNE comparing healthy, vaccinia-naïve men with women and suggest that sex should be considered in further development and deployment of IMVAMUNE and other MVA-based vaccines.     

Inactivated and live bivalent fowl adenovirus (FAdV8b + FAdV11) breeder vaccines provide broad-spectrum protection in chicks against inclusion body hepatitis (IBH)

Fowl adenovirus (FAdV) is comprised of five species (A to E) and 12 serotypes (1–7, 8a, 8b, 9–11). Inclusion body hepatitis (IBH) is caused by FAdV-7, 8a, 8b (species E) and FAdV-2 and 11 (species D). Commercial vaccines against IBH are not available in Canada. Autogenous FAdV broiler breeder vaccines are now used in some areas where outbreaks of IBH are occurring. The objective of this study was to evaluate the efficacy of a bivalent (species D and E) live and an inactivated FAdV broiler breeder vaccine in protecting broiler chicks against IBH through maternal antibody (MtAb) transfer. FAdV seronegative broiler breeders (n = 300/group) received either a live or inactivated bivalent (FAdV-8b-SK + FAdV-11-1047) vaccine. The live vaccine (1 × 10⁴ TCID₅₀ of each virus/bird) was given orally once at 16 weeks of age and the inactivated vaccine (1 × 10⁶TCID₅₀ of each virus + 20% Emulsigen D) was given intramuscularly at 16 and 19 weeks of age. Controls (n = 150) were given saline orally. The inactivated vaccine group was boosted 3 weeks later with the same vaccine. Neutralizing antibodies (NAb) in sera (n = 10) were detected at 19, 22, 30 and 48 weeks of age. NAb were able to neutralize various FAdV serotypes within species D and E. Mean NAb were similar in the both live and killed vaccine groups at 19, 30 and 48 weeks and ranged from 2.4 to 3.7 log₁₀. Approximately 26 ± 7% of MtAbs were passively transferred through eggs to day-old chicks. Progeny challenged with a lethal dose (1 × 10⁷ TCID₅₀/bird intramuscularly) of FAdV-8b-SK, FAdV-11-1047, or FAdV-2-685 (n = 90/group) at 14 days post-hatch (dph) showed 98–100% protection in broiler chicks to homologous or heterologous FAdV challenges. Our data suggests that a bivalent live and an inactivated FAdV vaccine are equally effective and have the potential for the control of IBH.     

Semi-perfusion cultures of suspension MDCK cells enable high cell concentrations and efficient influenza A virus production

Control and prevention of rapid influenza spread among humans depend on the availability of efficient and safe seasonal and pandemic vaccines, made primarily from inactivated influenza virus particles. Current influenza virus production processes rely heavily on embryonated chicken eggs or on cell culture as substrate for virus propagation. Today’s efforts towards process intensification in animal cell culture could innovate viral vaccine manufacturing using high-yield suspension cells in high cell density perfusion processes. In this work, we present a MDCK cell line adapted to grow as single cell suspension with a doubling time of less than 20 h, achieving cell concentrations over 1 × 10⁷ cells/mL in batch mode. Influenza A virus titer obtained in batch infections were 3.6 log₁₀(HAU/100 µL) for total- and 10⁹ virions/mL for infectious virus particles (TCID₅₀), respectively. In semi-perfusion mode concentrations up to 6 × 10⁷ cells/mL, accumulated virus titer of 4.5 log₁₀(HAU/100 µL) and infectious titer of almost 10¹⁰ virions/mL (TCID₅₀) were possible. This exceeds results reported previously for cell culture-based influenza virus propagation by far and suggests perfusion cultures as the preferred method in viral vaccine manufacturing.     

Changes in circulating lymphocytes and lymphoid tissue associated with vaccination of colostrum deprived calves

The objective of this study was to compare immunological responses and lymphoid depletion in young, colostrum deprived calves following administration of vaccines containing modified-live bovine viral diarrhea virus (BVDV). A group of calves exposed to a typical virulence non-cytopathic (ncp) BVDV-2 field strain (ncp exposed) was included to compare responses of calves receiving vaccine to responses generated against a field strain (mimicking a natural infection). A negative control group administered a placebo was used in all comparisons. All vaccines used in the study were administered per manufacturer recommendations while ncp BVDV exposed calves received 5 ml intranasally (2.5 ml/nare; 4.2 × 10⁶ TCID₅₀/ml) of the BVDV-2 field strain. Samples collected at each time point included nasal swabs for virus detection, blood samples for complete blood counts and detection of viremia, PBMCs for flow cytometric analysis, serum for virus neutralization titers, and thymus tissue at necropsy for evaluation of lymphoid depletion. A measurable neutralizing BVDV titer was observed for all treatment groups excluding the control animals, which remained negative during the study period. Virus shedding was only detected from the ncp vaccinated and ncp exposed calves. A decline from baseline was observed for peripheral lymphocyte and CD4⁺ cells for the groups receiving the adjuvanted cytopathic (cp) vaccine, the double deleted genetically modified (ddGM) vaccine, the ncp vaccine and ncp exposed calves, but not for the control group or groups receiving cp vaccines. Thymus depletion was observed for the ncp vaccine and ncp exposed calves and to a lesser extent for the ddGM vaccine calves. Collectively, these data suggest that the virus biotype, method of attenuation, presentation, and use of adjuvant will influence vaccine impacts on lymphoid tissues and the immune response. As such, multiple variables should be considered when determining costs and benefits of vaccination.     

Protective efficacy of a bivalent H5 influenza vaccine candidate against both clades 2.3.2.1 and 2.3.4.4 high pathogenic avian influenza viruses in SPF chickens

Worldwide, high pathogenic avian influenza viruses belonging to clades 2.3.4.4 and 2.3.2.1 have been circulating in both poultry and wild birds. Since 2018, Korea has built a national antigen bank to ensure preparedness in an emergency. In this study, we developed a bivalent vaccine candidate containing antigens derived from two reassortant KA435/2.3.2.1d and H35/2.3.4.4b strains for Korean national antigen bank. We evaluated its immunogenicity and protective efficacy in specific pathogen free chickens. The two vaccine strains, rgKA435-H9N2 PB2/2.3.2.1d and rgH35/2.3.4.4b, both of which were generated successfully by reverse genetics, were highly immunogenic (titres of haemagglutination inhibition: 8.3 and 8.4 log₂, respectively) and showed good protective efficacy (100 and 147 50% protective dose, respectively) against lethal challenge with wild-type virus when delivered as a 1:1 mixture. Notably, the vaccine provided complete protection against viral shedding at a full dose (512 HAU) and a 1/10 dose (51.2 HAU), with no clinical signs, after challenge with H35/2.3.4.4b. The bivalent vaccine developed in this study may reduce the cost of vaccine production and could be used as a H5 subtype avian influenza vaccine candidate against two clades simultaneously.     

Development and serology based efficacy assessment of a trivalent foot-and-mouth disease vaccine

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals throughout the world. The endemicity of this disease in Bangladesh has been causing high economic loss and an impediment to the full potential surge of livestock industries. In Bangladesh, vaccination using imported or locally produced FMD vaccines is the existing practice of controlling the disease, although vaccine failure cases are very common. Hence, to address the problem, the present study was envisaged to develop an effective FMD vaccine tailored to the circulating indigenous foot-and-mouth disease virus (FMDV) strains. Three local circulating FMDVs O/BAN/TA/Dh-301/2016 (MK088170.1), A/BAN/CH/Sa-304/2016 (MK088171.1) and Asia1/BAN/DH/Sa-318/2018 (MH457186.1) isolates were selected as vaccine strains based on recent epidemiology, genetic and antigenic analyses. These serotype O, A and Asia1 vaccine strains showed strong antigenic relationship (r1 > 0.3) with 100% to 75% of the respective circulating viruses. The candidate viruses were successfully inactivated by 3.0 mM binary ethylenimine within 7–10 h after the onset of inactivation. Extrapolation of inactivation kinetics confirmed < 1 log₁₀ TCID₅₀ in a 10000-liter batch liquid preparation after 24 h inactivation cycle. The inactivated virus particles were significantly (p < 0.05) concentrated and the trivalent vaccine was formulated using 6 µg per dose per serotype antigen payload. The trivalent vaccine was administered in divided doses in different groups of cattle. All doses of the vaccine elicited significantly (p < 0.05) higher levels of antibodies as early as 14-day post-vaccination (dpv) and peak antibody titers were achieved in 28 dpv. The ‘full dose’ (6.0 µg per dose per serotype) vaccine elicited antibody titers expected to confer protection in 100% cattle of the respective group and maintained such level of antibodies beyond 180 dpv. Thus, the trivalent FMD vaccine prepared with 6.0 µg antigen per dose per serotype of the selected candidate viruses will confer protection against circulating FMDVs of Bangladesh and its neighboring countries.     

Rubella specific cell-mediated and humoral immunity following vaccination in college students with low antibody titers

ObjectiveThis study measured cell-mediated immunity (CMI) and antibodies to clarify the basis of rubella reinfection after vaccination.MethodsIn a pool of 65 college students, 39 who exhibited hemagglutination–inhibition (HI) antibody titers against rubella of ≤1:16 were vaccinated with a rubella vaccine. The CMI was assessed with interferon-gamma release assay.ResultsThere was low correlation (r = 0.24) between the antibody titers and interferon-gamma levels at pre-vaccination status. Preexisting interferon-gamma levels were low in some subjects with low HI antibody titers of 1:8 and 1:16. Fifty-seven percent (4/7) of the subjects who were antibody-negative with past history of rubella vaccination at entry onto the study exhibited CMI. And 57% (4/7) of the subjects remained antibody-negative following a second vaccination, despite exhibiting CMI. HI antibody titers increased significantly after vaccination, whereas post-vaccination interferon-gamma levels did not exhibit significant increases. When subjects were divided (based on their past history of vaccination and antibody values) into natural infection and vaccination groups, HI antibody titers (mean ± SD) increased to 1:2^4.4 ± 1.4 from 1: 2^3.2 ± 0.4 (p = 0.065) in the natural infection group and to 1:2^4.4 ± 1.0 from 1:2^3.0 ± 0.8 (p < 0.00001) in the vaccination group following vaccination. The same classification revealed that interferon-gamma values did not increase significantly in either group following vaccination, but the interferon-gamma values at pre- and post-vaccination in the natural infection group were significantly higher than those at pre- and post-vaccination in the vaccination group (p < 0.05 and p < 0.05, respectively).ConclusionPre-vaccination interferon-gamma levels in each HI antibody titer group were similar. And there were some subjects with antibody-positive exhibited CMI-negative. These data may explain why rubella reinfection can occur in vaccinated seropositive individuals.     

Safety and immunogenicity of three tetravalent dengue vaccine formulations in healthy adults in the USA

Background

A candidate recombinant, live-attenuated, CYD tetravalent dengue vaccine (CYD-TDV) has recently demonstrated immunogenicity, efficacy and good tolerability. This study was performed to evaluate three CYD-TDV formulations in adults.

Methods

This was a randomized, double-blind, multicenter, phase II trial. The vaccine formulations were: CYD-TDV 5555 (≈5 log₁₀ tissue culture infectious dose 50% [TCID₅₀] of serotypes 1–4); CYD-TDV 5553 (≈5 log₁₀ TCID₅₀ of serotypes 1–3 and ≈3 log₁₀ TCID₅₀ of serotype 4); and CYD-TDV 4444 (≈4 log₁₀ TCID₅₀ of serotypes 1–4). Vaccinations were administered at 0, 6 and 12 months. Immunogenicity was assessed using the plaque reduction neutralization test.

Results

In total, 260 individuals were enrolled. The 5555 formulation elicited a superior serotype 4 response versus the 5553 formulation, with seropositivity rates of 89.7% and 58.3%, respectively, after the second dose (between-group difference 31.4%; 95% confidence interval 18.2–43.2). After each of the three doses, seropositivity rates for serotypes 1–3 were numerically highest with CYD-TDV 5553 and lowest with the 4444 formulation; seropositivity rates for serotype 4 were similar with the 5555 and 4444 formulations, and much lower among recipients of CYD-TDV 5553. Geometric mean titers followed the same pattern as that seen with seropositivity rates. Safety/reactogenicity results were similar for all three vaccine formulations, although the percentage of participants reporting solicited injection site reactions was lower with CYD-TDV 4444 than with the other two formulations. All serious adverse events were unrelated to vaccination.

Conclusions

Reducing the dose of serotype 4 antigen (5553 formulation) creates an imbalance in the immune response to CYD-TDV. Immune responses to CYD-TDV 5555 were slightly higher than to the 4444 formulation. Development of CYD-TDV 5555 has subsequently been pursued.     

Newcastle disease virus vectored infectious laryngotracheitis vaccines protect commercial broiler chickens in the presence of maternally derived antibodies

Newcastle disease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoproteins B and D have previously been demonstrated to confer complete clinical protection against virulent ILTV and NDV challenges in naive chickens. We extended this study to assess whether maternally derived antibody (MDA) against NDV and ILTV would interfere with protection in vaccinated broiler chickens. Chickens with a mean NDV MDA hemagglutination inhibition (HI) titer of 6.4 (log₂) and detectable ILTV neutralization (VN) antibodies at hatch were vaccinated with rLS/ILTV-gB or rLS/ILTV-gD at 1 or 10 day of age (DOA) or on both days. Groups of birds vaccinated with the commercial ILT vaccines (FP-LT and CEO) or sham inoculated were also included in this study. All vaccinated birds were challenged with virulent ILTV strain at 21 DOA. By that time, NDV HI titers declined to 2.6 (log₂) in unvaccinated birds, whereas the HI titers in NDV vectored vaccine groups increased to 3.5–6.3 (log₂). At standard dosages, both vaccine candidates conferred significant clinical protection; however, the protection elicited by the rLS/ILTV-gD was superior to that of rLS/ILTV-gB. Recombinant rLS/ILTV-gD reduced ILTV shedding from tracheal and ocular tissues by approximately 3 log₁₀ TCID₅₀. Notably, there was no improvement in protection after booster vaccination at 10 DOA. Overall results indicate that the presence of maternal antibodies to NDV and ILTV did not significantly interfere with the ability of the NDV LaSota strain-vectored ILTV gB and gD vaccine candidates to elicit protective immunity against infectious laryngotracheitis.     

Homologous and heterologous antigenic matched vaccines containing different H5 hemagglutinins provide variable protection of chickens from the 2014 U.S. H5N8 and H5N2 clade 2.3.4.4 highly pathogenic avian influenza viruses

From December 2014 to June 2015, a novel H5 Eurasian A/goose/Guangdong (Gs/GD) lineage clade 2.3.4.4 high pathogenicity avian influenza (HPAI) virus caused the largest animal health emergency in US history resulting in mortality or culling of greater than 48 million poultry. The outbreak renewed interest in developing intervention strategies, including vaccines, for these newly emergent HPAI viruses. In these studies, several existing H5 vaccines or vaccine seed strains with varying genetic relatedness (85–100%) to the 2.3.4.4 HPAI viruses were evaluated for protection in poultry. Chickens received a single dose of either an inactivated whole H5 AI vaccine, or a recombinant fowl poxvirus or turkey herpesvirus-vectored vaccines with H5 AI hemagglutinin gene inserts followed by challenge with either a U.S. wild bird H5N8 (A/gyrfalcon/Washington/40188-6/2014) or H5N2 (A/northern pintail/Washington/40964/2014) clade 2.3.4.4 isolate. Results indicate that most inactivated H5 vaccines provided 100% protection from lethal effects of H5N8 or H5N2 challenge. In contrast, the recombinant live vectored vaccines only provided partial protection which ranged from 40 to 70%. Inactivated vaccine groups, in general, had lower number of birds shedding virus and at lower virus titers then the recombinant vaccine groups. Interestingly, prechallenge antibody titers using the HPAI challenge viruses as antigen in heterologous vaccine groups were typically low (≤2 log₂), yet the majority of these birds survived challenge. Taken together, these studies suggest that existing vaccines when used in a single immunization strategy may not provide adequate protection in poultry against the 2.3.4.4 HPAI viruses. Updating the H5 hemagglutinin to be genetically closer to the outbreak virus and/or using a prime-boost strategy may be necessary for optimal protection.     

Antigenic variation of LaSota and genotype VII Newcastle disease virus (NDV) and their efficacy against challenge with velogenic NDV

Continued monitoring and evaluation of vaccine efficacy against prevalent or newly isolated strains has great importance in advising Newcastle disease (ND) immunization strategy. In this study, we systematically analysed the antigenic variation between genotype VII NDV aSG10 and the commercial vaccine strain LaSota, and assessed their efficacy against challenge with velogenic NDV by serological analysis and animal testing. We show that these two viruses are antigenically distinguishable; anti-NDV aSG10 hyper-immune sera demonstrated higher haemagglutination inhibition (HI) titres (11.13 ± 0.30log₂) against the aSG10 virus, compared with titres against LaSota (9.53 ± 0.50log₂). Conversely, the hyper-immune sera from LaSota showed higher HI titres against LaSota virus (9.73 ± 0.36log₂), but 2-fold lower HI titre against aSG10 (8.87 ± 0.38log₂). Each serum neutralised heterologous virus, but neutralisation titres were always 3- to 6-fold higher against its homologous strain than heterologous virus. The cross-reactivity R value between aSG10 and LaSota was 0.23, indicating that they are loosely related with major antigenic differences within a single serotype. The results of animal tests revealed that the aSG10 vaccine had a significantly higher protection rate than the LaSota vaccine against genotype VII NDV, regardless of intramuscular (IM) or eye drop/intranasal (ED/IN) route of SG10 challenge. Compared with IM administration, chicken flocks needed higher HI antibody levels to obtain sufficient protection when challenged by the natural ED/IN route. These results are highly informative for better control of ND in the poultry industry.     

Development of a live-attenuated influenza B ΔNS1 intranasal vaccine candidate

We discovered a unique, single amino acid mutation in the influenza B M1 protein promoting viral growth of NS1 truncation mutants in Vero cells. Due to this mutation, we were able to generate an influenza B virus lacking the complete NS1 open reading frame (ΔNS1-B virus) by reverse genetics, which was growing to titers of 8 log₁₀ TCID₅₀/ml in a Vero cell culture-based micro-carrier fermenter. The ΔNS1-B vaccine candidate was attenuated in IFN-competent hosts such as human alveolar epithelial cells (A549) similar to influenza A ΔNS1 viruses. In ferrets, the ΔNS1-B virus was replication-deficient and did not provoke any clinical symptoms. Importantly, a single intranasal immunization of ferrets at a dose as low as 6 log₁₀ TCID₅₀/animal induced a significant HAI response and provided protection against challenge with wild-type influenza B virus. So far, the lack of a ΔNS1-B virus component growing to high titers in cell culture has been limiting the possibility to formulate a trivalent vaccine based on deletion of the NS1 gene. Our study closes this gap and paves the way for the clinical evaluation of a seasonal, trivalent, live replication-deficient ΔNS1 intranasal influenza vaccine.     

Development of a high-growth enterovirus 71 vaccine candidate inducing cross-reactive neutralizing antibody responses

Although Enterovirus 71 (EV71) has only one serotype based on serum neutralization tests using hyperimmune animal antisera, three major genogroups (A, B and C) including eleven genotypes (A, B1-B2, and C1-C5) can be well classified based on phylogenetic analysis. Since 1997, large-scale EV71 epidemics occurred cyclically with different genotypes in the Asia-Pacific region. Therefore, development of EV71 vaccines is a national priority in several Asian countries. Currently, five vaccine candidates have been evaluated in clinical trials in China (three C4 candidates), Singapore (one B2 candidate), and Taiwan (one B4 candidate). Overall, the peak viral titers of these 5 vaccine candidates could only reach about 10⁷ TCID₅₀/mL. Moreover, genotypes of these 5 candidates are different from the current predominant genotype B5 in Taiwan and South-Eastern Asia. We adapted a high-growth EV71 genotype B5 (HG-B5) virus after multiple passages and plaque selections in Vero cells and the HG-B5 virus could reach high titers (>10⁸ TCID₅₀/mL) in a microcarrier-based cell culture system. The viral particles were further purified and formulated with alum adjuvant. After two doses of intramuscular immunization in rabbits, the HG-B5 vaccine candidate could induce cross-reactive neutralizing antibodies against the three major EV71 genogroups. In conclusion, a high-growth EV71 virus was successfully adapted in Vero cells and could induce broad spectrum neutralizing antibody titers against three (A, B5, and C4) genotypes in rabbits.     

Development of a high-dose vaccine formulation for prevention of megalocytivirus infection in rock bream (Oplegnathus fasciatus)

A formalin-inactivated red sea bream iridovirus (RSIV) vaccine was prepared using the culture supernatant of a persistently infected Pagrus major fin cell line (PI-PMF) with IVS-1 strain (RSIV subtype II Meglaocytivirus). Rock bream (Oplegnathus fasciatus) were injected with a high-dose, ultracentrifuged megalocytivirus vaccine (Ultra HSCMV, 7.0 × 10¹⁰ copies/mL), a high-dose supernatant of cultured megalocytivirus vaccine (HSCMV, 1.0 × 10¹⁰ copies/mL), a supernatant of cultured megalocytivirus vaccine (SCMV, 1.0 × 10⁹ copies/mL), and a low-dose of cultured megalocytivirus vaccine (LSCMV, 1.0 × 10⁸ copies/mL). The vaccine efficacies for the various vaccine formulations were determined done following injection challenge with IVS-1 (1.0 × 10⁴ copies/0.1 mL/fish), and the four different vaccines exhibited cumulative mortalities of 10.0 ± 0.0%, 48.3 ± 7.6%, 75.0 ± 5.0%, and 100.0 ± 0.0%, respectively. Additionally, the dose-dependent vaccine efficacy was also confirmed using two different cohabitation methods that included challenges G (general) and I (individual). When squalene + aluminum hydroxide (SqAl) was used as an adjuvant for the HSCMV or SCMV vaccine, cumulative mortalities of 30.0 ± 5.0% and 48.3 ± 7.6%, respectively, were obtained; moreover, these two adjuvants exhibited the highest efficacy in this study. The observed difference in survival post-challenge for the different vaccine concentrations was not reflected in the differences in neutralizing antibody titers. It was found that the water temperature during immune induction plays a less important a role than the water temperature during the challenge test, in which lowering the water temperature from 25 °C to 21 °C during a challenge improved the level of protection from cumulative mortalities from 35% to 10%. This study demonstrated that protection against mortality using inactivated vaccines against RSIVD in rock bream, which are known to be the most susceptible species to RSIV infection, is dependent upon antigen dose and temperature during the challenge.     

A single dose glycoprotein D-based subunit vaccine against pseudorabies virus infection

Pseudorabies Virus (PRV) is the causative agent of Pseudorabies (PR), also known as Aujeszky’s Disease, one of the most important infectious diseases in swine, resulting in huge economic losses to the swine industry globally. The emergence of mutant PRV strains after 2011 resulted in a sharp decrease in the efficacy of available commercial vaccines. To develop a more effective vaccine that can prevent the spread of PRV, glycoprotein B (gB), glycoprotein C (gC) and glycoprotein D (gD) from recent PRV isolates were expressed in a baculovirus system and their protective efficacy was tested in mice and piglets. Neutralizing antibody titers (NAs) in mice vaccinated with gB, gC and gD peaked at 28 days after immunization and then slowly declined. NAs in the mice immunized with gD were remarkably higher than other groups. After a lethal challenge of 5 LD₅₀ with mutant PRV-HNLH strain, the survival rates of gB and gD were 100% and 87.5% respectively, which was significantly higher than gC group (50%). Piglets vaccinated with the gD and gB + D vaccines developed the highest NAs 7 days post immunization. No piglets in these two groups exhibited clinical symptoms, high body temperature or virus shedding following challenge with 10^6.6 TCID₅₀ with the mutant PRV-HNLH strain. Histopathology and immunohistochemistry showed remarkably reduced pathological damage and viral loads in gD and gB + D groups. Furthermore, the duration of the NAs induced by gD vaccine could maintain as long as four months after a single dose. The current study indicates that a gD-based vaccine could be developed for the efficient control of PRV.