Deinococcus Mn2+-peptide complex: A novel approach to alphavirus vaccine development

Over the last ten years, Chikungunya virus (CHIKV), an Old World alphavirus has caused numerous outbreaks in Asian and European countries and the Americas, making it an emerging pathogen of great global health importance. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, on the other hand, has been developed as a bioweapon in the past due to its ease of preparation, aerosol dispersion and high lethality in aerosolized form. Currently, there are no FDA approved vaccines against these viruses.In this study, we used a novel approach to develop inactivated vaccines for VEEV and CHIKV by applying gamma-radiation together with a synthetic Mn-decapeptide-phosphate complex (MnDpPi), based on manganous-peptide-orthophosphate antioxidants accumulated in the extremely radiation-resistant bacterium Deinococcus radiodurans. Classical gamma-irradiated vaccine development approaches are limited by immunogenicity-loss due to oxidative damage to the surface proteins at the high doses of radiation required for complete virus-inactivation. However, addition of MnDpPi during irradiation process selectively protects proteins, but not the nucleic acids, from the radiation-induced oxidative damage, as required for safe and efficacious vaccine development. Previously, this approach was used to develop a bacterial vaccine. In the present study, we show that this approach can successfully be applied to protecting mice against viral infections.Irradiation of VEEV and CHIKV in the presence of MnDpPi resulted in substantial epitope preservation even at supra-lethal doses of gamma-rays (50,000 Gy). Irradiated viruses were found to be completely inactivated and safe in vivo (neonatal mice). Upon immunization, VEEV inactivated in the presence of MnDpPi resulted in drastically improved protective efficacy. Thus, the MnDpPi-based gamma-inactivation approach described here can readily be applied to developing vaccines against any pathogen of interest in a fast and cost-effective manner.     

Viable spores of Coccidioides posadasii Δcps1 are required for vaccination and provide long lasting immunity

Coccidioidomycosis is a systemic fungal infection for which a vaccine has been sought for over fifty years. The avirulent Coccidioides posadasii strain, Δcps1, which is missing a 6 kb gene, showed significant protection in mice. These studies explore conditions of protection in mice and elucidate the immune response. Mice were vaccinated with different doses and viability states of Δcps1 spores, challenged with virulent C. posadasii, and sacrificed at various endpoints, dependent on experimental objectives. Tissues from vaccinated mice were harvested for in vitro elucidation of immune response. Vaccination with viable Δcps1 spores was required for protection from lethal challenge. Viable spore vaccination produced durable immunity, lasting at least 6 months, and prolonged survival (≥6 months). The C. posadasii vaccine strain also protected mice against C. immitis (survival ≥ 6 months). Cytokines from infected lungs of vaccinated mice in the first four days after Cp challenge showed significant increases of IFN-γ, as did stimulated CD4⁺ spleen cells from vaccinated mice. Transfer of CD4⁺ cells, but not CD8⁺ or B cells, reduced fungal burdens following challenge. IFN-γ from CD4⁺ cells in vaccinated mice indicates a Th1 response, which is critical for host control of coccidioidomycosis.     

M2SR,a novel live influenza vaccine,protects mice and ferrets against highly pathogenic avian influenza

The emergence of highly pathogenic avian influenza H5N1 viruses has heightened global concern about the threat posed by pandemic influenza. To address the need for a highly effective universal influenza vaccine, we developed a novel M2-deficient single replication (M2SR) influenza vaccine virus and previously reported that it provided strong heterosubtypic protection against seasonal influenza viruses in mice. In the current study, we assessed M2SR induced protection against H5N1 influenza in mice and ferrets.Mice were intranasally inoculated with M2SR viruses containing the HA and NA from A/Vietnam/1203/2004 (M2SR H5N1) or A/California/07/2009 (M2SR H1N1). All M2SR vaccinated mice survived lethal challenge with influenza A/Vietnam/1203/2004 (H5N1), whereas 40% of mice vaccinated with recombinant H5 HA and none of the naïve controls survived. M2SR H5N1 provided sterile immunity, whereas low levels of virus were detected in the lungs of some M2SR H1N1 vaccinated mice. In contrast, recombinant H5 HA vaccinated mice and naïve controls showed systemic infection.M2SR H5N1 induced strong serum and mucosal antibody responses (IgG and IgA classes) against H5 HA, with high hemagglutination inhibition (HAI) titers. In contrast, while M2SR H1N1 elicited cross-reactive antibodies recognizing the H5 HA2 stalk region or the neuraminidase, no HAI activity against H5N1 virus was detected after M2SR H1N1 immunization.Both M2SR H5N1 and H1N1 also protected ferrets against lethal challenge with A/Vietnam/1203/2004. A prime–boost regimen provided optimal protection with no virus detected in the respiratory tract or brain after challenge. As in the mouse model, only the M2SR H5N1 vaccine induced HAI antibodies against the challenge virus in ferrets, while the M2SR H1N1 was able to provide protection without the induction of HAI antibodies.In summary, effective protection against highly pathogenic H5N1 influenza virus was provided by both homologous H5N1 M2SR and heterologous H1N1 M2SR demonstrating the cross-protective attributes of the M2SR platform.     

Rescue of CD8+ T cell vaccine memory following sublethal γ irradiation

Sublethal γ irradiation eliminates CD8+ T cell mediated memory responses. In this work, we explored how these memory responses could be rescued in the aftermath of such exposure. We utilized two models of CD8+ T cell mediated immunity: a mouse model of Listeria monocytogenes (LM) infection in which CD8+ T cells specific for LM expressed antigens (Listeriolysin O, LLO) can be tracked, and a murine skin graft model in which CD8+ T cells mediate rejection across a MHC class I (D^d) disparity. In the LM immunized mice, LL0 specific CD8+ T memory cells were lost on irradiation, preserved with rapid revaccination with an attenuated strain 1–3 days post-irradiation (PI), and these mice survived a subsequent wild type LM challenge. A genetic “signature of rescue” identified a group of immune-associated mRNA maintained or upregulated following irradiation and rescue. A number of these factors, including IL-36γ, dectin-2 (Clec4n), and mir101c are upregulated rapidly after exposure of mice to sublethal γ radiation alone and are sustained by early, but not later rescue. Such factors will be evaluated as potential therapeutics to replace individual vaccines for global rescue of CD8+ T memory cell responses following sublethal γ irradiation. The skin allograft model mirrored that of the LM model in that the accelerated D^d skin allograft rejection response was lost in mice exposed to sublethal γ radiation, but infusion of allogeneic D^d expressing bone marrow cells 1–4 days PI preserved the CD8+ T memory mediated accelerated rejection response, further suggesting that innate immune responses may not always be essential to rescue of CD8+ memory T cells following γ irradiation.     

Manipulation of neuraminidase packaging signals and hemagglutinin residues improves the growth of A/Anhui/1/2013 (H7N9) influenza vaccine virus yield in eggs

In 2013, a novel avian-origin H7N9 influenza A virus causing severe lower respiratory tract disease in humans emerged in China, with continued sporadic cases. An effective vaccine is needed for this virus in case it acquires transmissibility among humans; however, PR8-based A/Anhui/1/2013 (Anhui/1, H7N9), a WHO-recommended H7N9 candidate vaccine virus (CVV) for vaccine production, does not replicate well in chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we explored the possibility that PR8’s hemagglutinin (HA) and neuraminidase (NA) packaging signals mediate improvement of Anhui/1 CVV yield in eggs. We constructed chimeric HA and NA genes having the coding region of Anhui/1 HA and NA flanked by the 5′ and 3′ packaging signals of PR8’s HA and NA, respectively. The growth of CVVs containing the chimeric HA was not affected, but that of those containing the chimeric NA gene grew in embryonated chicken eggs with a more than 2-fold higher titer than that of WT CVV. Upon 6 passages in eggs further yield increase was achieved although this was not associated with any changes in the chimeric NA gene. The HA of the passaged CVV, did, however, exhibit egg-adaptive mutations and one of them (HA-G218E) improved CVV growth in eggs without significantly changing antigenicity. The HA-G218E substitution and a chimeric NA, thus, combine to provide an Anhui/1 CVV with properties more favorable for vaccine manufacture.     

Enhanced immune responses to E2 protein and DNA formulated with ISA 61 VG administered as a DNA prime–protein boost regimen against bovine viral diarrhea virus

The aim of this study was to develop and test an optimal vaccination strategy against bovine viral diarrhea virus (BVDV) based on the E2 glycoprotein of the BJ1305 strain. To achieve higher E2-specific antibody titers and to broaden the cellular immune response, a plasmid encoding the E2 protein (pcDNA3.1-E2) was constructed and a purified recombinant E2 protein was generated. The E2 protein was emulsified in the adjuvant ISA 61 VG prior to administration. We immunized mice three times with pcDNA3.1-E2 or the recombinant E2 protein or primed twice with pcDNA3.1-E2 and boosted once with the E2 protein. To evaluate the protection against BVDV conferred by the vaccines, the mice were challenged with BVDV strain Oregon C24V after the third immunization. Although all immunized mice developed humoral and cellular immune responses, the E2-specific antibody titers in the DNA prime–protein boost group were significantly higher than those elicited by either the DNA or the protein vaccine. In addition, vaccination with the E2 DNA vaccine induced higher percentages of CD4⁺IFN-γ⁺ T cells and CD8⁺IFN-γ⁺ T cells among total CD3⁺ T cells than the other regimens. The predominant antibody subclass in the vaccinated mice was IgG1. Serum tumor necrosis factor alpha (TNF-α) levels in the DNA prime–protein boost group were significantly higher after the third immunization than in the other groups. Moreover, the mice treated with the DNA prime–protein boost vaccination regimen acquired protection against BVDV challenge, as shown by a significant reduction of viremia, only minor pathological changes, and a lower viral antigen burden than in the control and solo vaccinated mice. These results demonstrate the potential advantage of a DNA prime–protein boost vaccination approach over a solo vaccination for the prevention of BVDV. The ability of this vaccine strategy to control and eradicate BVD in herds warrants further investigation.     

Development of a self-assembling protein nanoparticle vaccine targeting Plasmodium falciparum Circumsporozoite Protein delivered in three Army Liposome Formulation adjuvants

We have developed FMP014, a vaccine candidate against Plasmodium falciparum malaria, which is comprised of 60 identical monomer protein chains that form an icosahedral shaped self-assembling protein nanoparticle (SAPN). Each monomer contains selected P. falciparum Circumsporozoite Protein (PfCSP) CD4+ and CD8+ epitopes, universal T_H epitopes, portions of the α-TSR domain, and 6 repeats of the NANP motifs of the PfCSP. Here we describe the conditions that are required for successful scale-up and cGMP manufacturing of FMP014 with a yield of ≈1.5 g of drug substance per 100 g of wet bacterial paste. When adjuvanted with an Army Liposomal Formulation (ALF) based adjuvant, the nanoparticle vaccine is highly immunogenic and prevents infection of mice by an otherwise lethal dose of transgenic P. berghei sporozoites expressing the full-length PfCSP.     

Optimizing seroprotection against pneumococcus in children with nephrotic syndrome using the 13-valent pneumococcal conjugate vaccine

BackgroundInfections are among the main life-threatening complications in patients with nephrotic syndrome (NS), in particular with Streptococcus pneumoniae, the first cause of bacterial peritonitis and sepsis in these patients. This study aims to evaluate the baseline seroprotection of NS patients against S. pneumoniae, and immunize them with the 13-valent pneumococcal conjugate vaccine (PCV13) regardless of disease activity and previous immunization history, in order to evaluate the immunogenicity, safety profile, and effect of NS treatment on vaccine responses.MethodsThis multicentre prospective interventional study enrolled 42 children with NS at disease onset or during a regular follow-up appointment. PCV13 was administered at inclusion. Serotype-specific S. pneumoniae IgG titer were assessed at baseline, after immunization, and at 1 year follow-up. Vaccine safety was evaluated clinically and by urinary tests.ResultsPCV13 induced high serotype-specific IgG titers that were maintained at high levels one year after vaccination, even in children previously immunized. No serious adverse event occurred and relapse frequency was unchanged.ConclusionGiven that high IgG titers were achieved and maintained after PCV13 vaccination, and considering the high morbidity related to S. pneumoniae, we propose PCV13 (re-)vaccination for all NS patients, irrespective of their previous immunization history, treatment and disease activity.     

The Plasmodium yoelii microgamete surface antigen (PyMiGS) induces anti-malarial transmission blocking immunity that reduces microgamete motility/release from activated male gametocytes

Malaria transmission-blocking vaccines aim to inhibit the development of malaria parasites in mosquitoes by inducing antibodies targeting surface proteins of sexual stage parasites. We have recently identified PyMiGS, a protein specifically expressed in the osmiophilic body of male gametocytes of Plasmodium yoelii (Py). PyMiGS is translocated to the surface of microgametes, and potent transmission-blocking activity was observed in mosquitoes fed on mice passively immunized with antibodies against PyMiGS. Here we demonstrate using a direct feeding assay that recombinant PyMiGS successfully induces anti-PyMiGS antibodies in mice and that the antibodies block parasite development in mosquitoes. We also show using the membrane-feeding assay that rabbit anti-PyMiGS antibody inhibits parasite development in mosquitoes in a dose-dependent manner without complement involvement. To investigate the mode of action of anti-PyMiGS antibodies against parasite development, we observed exflagellation after mixing Py gametocytes with activation medium containing anti-PyMiGS or anti-GST control antibodies. Whereas most microgametes were released from activated male gametocytes in the control group, a significantly reduced number of microgametes were released in the anti-PyMiGS group, with most of the microgametes left attached to the activated male gametocytes. Moreover, anti-PyMiGS antibodies shortened the duration of the active movement of microgametes after the onset of exflagellation. Taken together, these findings suggest that anti-PyMiGS antibodies bind to the microgamete surface immediately after exflagellation, thereby reducing microgamete motility and inhibiting microgamete release from the activated male gametocytes. These results strongly suggest that PyMiGS orthologues in Plasmodium falciparum and Plasmodium vivax can be promising TBV candidates.     

Intranasal immunization with immunodominant epitope peptides derived from HpaA conjugated with CpG adjuvant protected mice against Helicobacter pylori infection

HpaA is considered to be an effective protective antigen for vaccination against Helicobacter pylori (H. pylori) infection. Oral immunization with HpaA significantly decreases bacterial colonization in H. pylori infected mice. In this study, we investigated whether subcutaneous or intranasal immunization with HpaA could protect against H. pylori infection. Mice immunized subcutaneously with HpaA in Complete Freund’s adjuvant, but not mice intranasally immunized with HpaA in CpG adjuvant acquired protection against H. pylori infection. However, intranasal immunization with immunodominant epitope peptides in CpG adjuvant protected mice against H. pylori infection, and immunodominant epitope peptides stimulated stronger Th1 responses and mediated more robust protection against H. pylori infection than subdominant ones. Our results suggest that the length of a candidate antigen is critical for particular vaccination routes, and that immunodominant epitope peptides are promising candidates for protection against H. pylori infection through nasal vaccination.     

Combined semi-empirical screening and design of experiments (DOE) approach to identify candidate formulations of a lyophilized live attenuated tetravalent viral vaccine candidate

A combination experimental approach, utilizing semi-empirical excipient screening followed by statistical modeling using design of experiments (DOE), was undertaken to identify stabilizing candidate formulations for a lyophilized live attenuated Flavivirus vaccine candidate. Various potential pharmaceutical compounds used in either marketed or investigative live attenuated viral vaccine formulations were first identified. The ability of additives from different categories of excipients, either alone or in combination, were then evaluated for their ability to stabilize virus against freeze-thaw, freeze-drying, and accelerated storage (25 °C) stresses by measuring infectious virus titer. An exploratory data analysis and predictive DOE modeling approach was subsequently undertaken to gain a better understanding of the interplay between the key excipients and stability of virus as well as to determine which combinations were interacting to improve virus stability. The lead excipient combinations were identified and tested for stabilizing effects using a tetravalent mixture of viruses in accelerated and real time (2–8 °C) stability studies. This work demonstrates the utility of combining semi-empirical excipient screening and DOE experimental design strategies in the formulation development of lyophilized live attenuated viral vaccine candidates.     

Development and application of a bioluminescent imaging mouse model for Chikungunya virus based on pseudovirus system

Chikungunya virus (CHIKV) is an arthropod-borne virus that is transmitted to humans primarily via the bite of an infected mosquito. Infection of humans by CHIKV can cause chikungunya fever which is an acute febrile illness associated with severe, often debilitating polyarthralgias. Since a re-emergence of CHIKV in 2004, the virus has spread into novel locations in nearly 40 countries including non-endemic regions and has led to millions of cases of disease throughout countries. Handling of CHIKV is restricted to the high-containment Biosafety Level 3 (BSL-3) facilities, which greatly impede the research progress of this virus. In this study, an envelope-pseudotyped virus expressing the firefly luciferase reporter protein (pHIV–CHIKV–Fluc) was generated. An in vitro sensitive neutralizing assay and an in vivo bioluminescent-imaging-based mouse infection model had been developed based on the CHIKV pseudovirus. Utilizing the platform, protection effect of DNA vaccine was evaluated. Therefore, this study provides a safe, sensitive and visualizing model for evaluating vaccines and antiviral therapies against CHIKV in low containment BSL-2 laboratories.     

Immunization of preterm infants with GSK’s hexavalent combined diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated poliovirus-Haemophilus influenzae type b conjugate vaccine: A review of safety and immunogenicity

BackgroundInfants with history of prematurity (<37 weeks gestation) and low birth weight (LBW, <2500 g) are at high risk of infection due to functional immaturity of normal physical and immunological defense mechanisms. Despite current recommendations that infants with history of prematurity/LBW should receive routine immunization according to the same schedule and chronological age as full-term infants, immunization is often delayed.MethodsHere we summarize 10 clinical studies and 15 years of post-marketing safety surveillance of GSK’s hexavalent vaccine (DTPa-HBV-IPV/Hib), a combined diphtheria-tetanus-acellular-pertussis-hepatitis-B-inactivated-poliovirus-Haemophilus influenzae-type-b (Hib) conjugate vaccine, when administered alone, or co-administered with pneumococcal conjugate, rotavirus, and meningococcal vaccines and respiratory syncytial virus IgG to infants with history of prematurity/LBW in clinical trials.ResultsAt least 92.5% of infants with history of prematurity/LBW as young as 24 weeks gestation in clinical studies were seropositive to all vaccine antigens after 3-dose primary vaccination with GSK’s hexavalent DTPa-HBV-IPV/Hib vaccine, with robust immune responses to booster vaccination. Seropositivity rates and antibody concentrations to hepatitis B and Hib appeared lower in infants with history of prematurity/LBW than term infants. Between 13–30% of medically stable infants with history of prematurity developed apnea after vaccination with GSK’s hexavalent DTPa-HBV-IPV/Hib vaccine; usually after dose 1. The occurrence of post-immunization cardiorespiratory events appears to be influenced by the severity of any underlying neonatal condition. Most cardiorespiratory events resolve spontaneously or require minimal intervention. GSK’s hexavalent DTPa-HBV-IPV/Hib vaccine was well tolerated in co-administration regimens.ConclusionGSK’s hexavalent DTPa-HBV-IPV/Hib vaccine alone or co-administered with other pediatric vaccines has a clinically acceptable safety and immunogenicity profile when used in infants with history of prematurity/LBW for primary and booster vaccination. Additional studies are needed in very premature and very LBW infants. However, currently available data support using GSK’s hexavalent DTPa-HBV-IPV/Hib vaccine to immunize infants with history of prematurity/LBW according to chronological age.     

Neutralising antibody response in domestic cats immunised with a commercial feline immunodeficiency virus (FIV) vaccine

Across human and veterinary medicine, vaccines against only two retroviral infections have been brought to market successfully, the vaccines against feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV). FeLV vaccines have been a global success story, reducing virus prevalence in countries where uptake is high. In contrast, the more recent FIV vaccine was introduced in 2002 and the degree of protection afforded in the field remains to be established. However, given the similarities between FIV and HIV, field studies of FIV vaccine efficacy are likely to advise and inform the development of future approaches to HIV vaccination.Here we assessed the neutralising antibody response induced by FIV vaccination against a panel of FIV isolates, by testing blood samples collected from client-owned vaccinated Australian cats. We examined the molecular and phenotypic properties of 24 envs isolated from one vaccinated cat that we speculated might have become infected following natural exposure to FIV. Cats vaccinated against FIV did not display broadly neutralising antibodies, suggesting that protection may not extend to some virulent recombinant strains of FIV circulating in Australia.     

Polyfunctional CD4 T-cells correlate with in vitro mycobacterial growth inhibition following Mycobacterium bovis BCG-vaccination of infants

BackgroundVaccination with Bacillus Calmette Guerin (BCG) protects infants against childhood tuberculosis however the immune mechanisms involved are not well understood. Further elucidation of the infant immune response to BCG will aid with the identification of immune correlates of protection against tuberculosis and with the design of new improved vaccines. The purpose of this study was to investigate BCG-induced CD4+ T-cell responses in blood samples from infants for cytokine secretion profiles thought to be important for protection against tuberculosis and compare these to PBMC-mediated in vitro mycobacterial growth inhibition.MethodsBlood from BCG-vaccinated or unvaccinated infants was stimulated overnight with Mycobacterium tuberculosis (M. tb) purified protein derivative (PPD) or controls and intracellular cytokine staining and flow cytometry used to measure CD4+ T-cell responses. PBMC cryopreserved at the time of sample collection were thawed and incubated with live BCG for four days following which inhibition of BCG growth was determined.ResultsPPD-specific IFNγ+TNFα+IL-2+CD4+ T-cells represented the dominant T-cell response at 4 months and 1 year after infant BCG. These responses were undetectable in age-matched unvaccinated infants. IL-17+ CD4+ T-cells were significantly more frequent in vaccinated infants at 4 months but not at 1-year post-BCG. PBMC-mediated inhibition of mycobacterial growth was significantly enhanced at 4 months post-BCG as compared to unvaccinated controls. In an analysis of all samples with both datasets available, mycobacterial growth inhibition correlated significantly with the frequency of polyfunctional (IFNγ+TNFα+IL-2+) CD4+ T-cells.ConclusionsThese data suggest that BCG vaccination of infants induces specific polyfunctional T-helper-1 and T-helper-17 responses and the ability, in the PBMC compartment, to inhibit the growth of mycobacteria in vitro. We also demonstrate that polyfunctional T-helper-1 cells may play a role in growth inhibition as evidenced by a significant correlation between the two.     

Temperature-mediated recombinant anthrax protective antigen aggregate development: Implications for toxin formation and immunogenicity

Anthrax vaccines containing recombinant PA (rPA) as the only antigen face a stability issue: rPA forms aggregates in solution after exposure to temperatures ⩾40 °C, thus losing its ability to form lethal toxin (LeTx) with Lethal Factor. To study rPA aggregation’s impact on immune response, we subjected rPA to several time and temperature combinations. rPA treated at 50 °C for 30 min formed high mass aggregates when analyzed by gel electrophoresis and failed to form LeTx as measured by a macrophage lysis assay (MLA). Aggregated rPA-formed LeTx was about 30 times less active than LeTx containing native rPA. Mice immunized with heat-treated rPA combined with Al(OH)₃ developed antibody titers about 49 times lower than mice immunized with native rPA, as measured by a Toxicity Neutralization Assay (TNA). Enzyme Linked Immunosorbent Assay (ELISA) of the same immune sera showed anti-rPA titers only 2–7 times lower than titers elicited by native rPA. Thus, rPA’s ability to form LeTx correlates with its production of neutralizing antibodies, and aggregation significantly impairs the protein’s antibody response. However, while these findings suggest MLA has some value as an in-process quality test for rPA in new anthrax vaccines, they also confirm the superiority of TNA for use in vaccine potency.     

Fibroblast-stimulating lipopeptide-1 as a potential mucosal adjuvant enhances mucosal and systemic immune responses to enterovirus 71 vaccine

To prevent viral infection at the site of entry, mucosal vaccines are potent tools for inducing IgA secretion for defense. Because Toll-like receptor (TLR) ligands serve as strong adjuvants, two ligands that mimic the structure of mycoplasmal and bacterial lipopeptides represent interesting vaccine candidates. Pam3CSK4, a synthetic triacylated lipopeptide, interacts with TLR2/1. Because fibroblast-stimulating lipopeptide-1 (FSL-1), a synthetic diacylated lipopeptide, is recognized by TLR2/6, we targeted the potential immuno-inducibility of Pam3CSK4 and FSL-1 as adjuvants of an enterovirus 71 (EV71) mucosal vaccine. Naïve BALB/c mice were used for intranasal immunization three times over a 3-week interval, with results showing that EV71-specific IgG and IgA in serum, nasal washes, bronchoalveolar lavage fluid, and feces from the EV71 + FSL-1 group were significantly higher than levels observed in mice treated with EV71 + Pam3CSK4, EV71 alone, or the control group treated with phosphate-buffered saline. Furthermore, we observed more EV71-specific IgG and IgA-producing cells in treatments using EV71 formulated with FSL-1. Additionally, T cell-proliferative responses and interferon-γ and interleukin-17 secretion were significantly increased when inactivated EV71 was formulated using FSL-1. Moreover, serum from immunized mice was capable of neutralizing the infectivity of EV71 (C2 genotype) and was able to cross-neutralize the B4 and B5 genotypes of EV71. Our data suggested that FSL-1 could be used as an efficient adjuvant for intranasal EV71-vaccine immunization.     

Inactivated influenza vaccine stress can affect in vitro potency assay relationship to immunogenicity

Influenza vaccines are the most effective intervention to prevent the substantial public health burden of seasonal and pandemic influenza. The capability of hemagglutinin (HA), the main antigen in inactivated influenza vaccines (IIVs), to elicit functional neutralizing antibodies determines IIV effectiveness. When HA is subjected to environmental stress during manufacturing or while stored prior to administration, such as low pH and temperature excursions, the HA immunological activity can be affected. Single-radial immunodiffusion (SRID), the standard in vitro potency assay for IIVs, is believed to specifically detect immunologically active HA and has been applied to evaluate HA stability against stress. Here we report that transient low pH treatment and freeze/thaw cycles with HA in PBS abolish SRID-quantified in vitro potency for all HAs of multiple influenza strains. Raised temperature substantially decreases in vitro potency with more extensive HA structural changes. Chemical stress and mechanical stress moderately change SRID in vitro potency values in a strain-dependent manner. Trypsin digestion, which selectively degrades stressed HA, followed by RP-HPLC quantification as a candidate alternative in vitro potency assay yields results comparable to SRID. Mouse immunogenicity studies confirm that HA stressed by transient low pH treatment does not elicit functional antibodies in vivo, nor does it have a measureable SRID value. However, HA stressed by raised temperature elicits high titers of functional antibodies in vivo despite substantial loss of SRID in vitro potency. This discrepancy between SRID in vitro potency and vaccine immunogenicity suggests that SRID may not reliably indicate IIV potency under all conditions. Further efforts to develop alternate potency assays that can better predict in vivo immunogenicity should continue along with additional studies exploring HA conformation, SRID values and consequent immunogenicity.     

Pertussis Immunisation in Pregnancy Safety (PIPS) Study: A retrospective cohort study of safety outcomes in pregnant women vaccinated with Tdap vaccine

BackgroundNew Zealand has funded the administration of tetanus, diphtheria and acellular pertussis (Tdap) vaccine during pregnancy to prevent infant pertussis since 2013. The aim of this study was to assess the safety of Tdap vaccine administered to pregnant women as part of a national maternal immunisation programme.MethodsWe conducted a national retrospective observational study using linked administrative New Zealand datasets. The study population consisted of pregnant women eligible to receive funded Tdap vaccination from 28 to 38 weeks gestation in 2013. Primary study outcomes were based on prioritised adverse events for the assessment of vaccine safety in pregnant women, as defined by WHO and Brighton Collaboration taskforces. We examined the effect of Tdap vaccination on prioritised maternal outcomes using Cox proportional hazard models. Adjusted hazard ratios controlled for key confounding variables.ResultsIn the cohort of 68,550 women eligible to receive funded antenatal Tdap vaccination during 2013, 8178 (11.9%) were vaccinated and 60,372 (88.1%) were unvaccinated. The use of Tdap in pregnancy was not associated with an increase in the rate of primary outcomes, including preterm labour; pre-eclampsia; pre-eclampsia with severe features; eclampsia; gestational hypertension; fetal growth restriction; or post-partum haemorrhage. Tdap also did not increase secondary outcomes, including gestational diabetes mellitus; antenatal bleeding; placental abruption; premature rupture of membranes; preterm delivery; fetal distress; chorioamnionitis; or, maternal fever during or after labour. Lactation disorders was the only secondary maternal outcome with a significantly increased hazard ratio. Tdap vaccine had a protective effect on pre-eclampsia with severe features, preterm labour, preterm delivery, and antenatal bleeding.ConclusionWe did not detect any biologically plausible adverse maternal outcomes following Tdap vaccination during pregnancy. This study provides further assurance that Tdap administration during pregnancy is not associated with unexpected safety risks.     

A novel challenge model to evaluate the efficacy of hepatitis C virus vaccines in mice

An effective hepatitis C virus (HCV) vaccine should elicit robust humoral and cell mediated immunity (CMI). A small animal challenge model is required to assess the efficacy of vaccines which elicit CMI. In this study, HCV proteins were expressed in hepatocytes of immunocompetent mice after hydrodynamic injection of a plasmid encoding the HCV NS3/4A protein. This vector, constructed as the “challenge”, was optimized for long term, specific gene expression in hepatocytes. To monitor HCV antigen expression in transfected hepatocytes, the plasmid also encoded secreted alkaline phosphatase (SEAP), which was detected in the mouse serum. The design of this novel challenge plasmid was based on studies using luciferase and SEAP as reporter molecules to examine the kinetics of the proteins expressed in hepatocytes and secreted into blood. We designed two constructs to control SEAP expression. In one construct, SEAP expression was controlled by the EMCV IRES, while in the other, a SEAP and luciferase polyprotein was cleaved by the FMDV2A proteinase. We found that SEAP expressed after FMDV2A self cleavage was more sensitive and showed a higher correlation with luciferase expressed in liver. The NS3/4A challenge model using the FMDV2A design provided a window period of 50 days to monitor changes in SEAP expression after hydrodynamic injection of DNA. In a challenge experiment, mice which received an adenovirus-based HCV vaccine showed accelerated clearance of SEAP and thus, of NS3/4A positive hepatocytes compared with a mock vaccinated group, that coincided with an increased number of CD8⁺ lymphocytes in the liver.