[Co-expression, purification and bioassay of three avian viral antigens].

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.

Human respiratory syncytial virus F protein expressed in Pichia pastoris or Escherichia coli induces protective immunity without inducing enhanced respiratory disease in mice.

Human respiratory syncytial virus (hRSV) is the primary cause of severe respiratory tract disease in children and infants as well as in elderly and immunocompromised adults. The fusion protein (F) of hRSV is the major antigen eliciting a neutralizing antibody response and protective immunity in the host, especially those recognizing the prefusion F protein (pre-F). In this study, we made genetic constructs for expression of a recombinant prefusion F protein in Pichia pastoris GS115, called RGF. Using Escherichia coli BL21, we expressed the pre-F and postfusion F protein (Post-F), called RBF and Post-RBF, respectively. RGF and RBF showed high affinity for 5C4, a highly potent monoclonal antibody specific for pre-F. We studied the immunogenicity of RGF and RBF in mice. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with RGF or RBF exhibited superior protective immunity, which was confirmed by serum neutralizing activity and viral clearance after challenge. As judged from the IgG1/IgG2a ratios and numbers of IFN-γ- and IL-4-secreting cells, RGF or RBF with alum adjuvant induced a balanced Th1-biased immune response and produced no signs of enhanced respiratory disease (ERD) upon hRSV challenge. In addition, the immunogenicity and protective efficacy of RGF were superior to those of RBF in mice. Therefore, RGF represents a potential vaccine candidate for the prevention of human infection with hRSV.

Establishment of an induced pluripotent cell line from Taiwan black silkie chick embryonic fibroblasts for replication-incompetent virus production.

The objective of this study was to establish a versatile cell line for replication-incompetent virus production and inactivation with formaldehyde to generate a model of cell-based vaccine manufacturing process. To achieve this goal, we took advantage of the easily accessed chick embryonic fibroblasts. Nine-day old chick embryonic fibroblasts were obtained and subjected to be transduced with a set of lentivirus to develop a chick induced pluripotent stem (ciPS) cell line. Morphological features, positive periodic acid-Schiff staining as well as strong immunocytofluorescence of alkaline phosphatase, intestinal (ALPI) and POU class 5 homeobox 1 (POU5F1) proteins suggested that these chick embryonic fibroblasts have been transformed into ciPS cells. Further differentiation and immunocytofluorescence assays confirmed that this ciPS cell line possesses capacities and potentials to form embryoid bodies, differentiate into all three embryonic layers: ectoderm, mesoderm and endoderm with evidence of strongly positive and specific molecular markers. Immunoblot analysis next demonstrated that through recombinant DNA technology and the 2nd generation lentiviral transfer system, the goose hemagglutinin gene (H5) gene was packaged into the replication-incompetent virus and highly expressed in a bladder cancer-derived cell line, T24, after transduction. The titer of ciPS-generated replication-incompetent virus is comparable to that from the Phoenix-AMPHO cell line, which is a commercial and high productive retrovirus producer. Our study successfully established a ciPS cell line which is able to produce replication-incompetent virus, providing a new strategy for cell-based vaccine production after virus inactivation.

Pichia pastoris-expressed Zika virus envelope domain III on a virus-like particle platform: design, production and immunological evaluation.

Zika virus (ZIKV) is an arbovirus which shares antigenic similarity and the mosquito vector with dengue viruses (DENVs). ZIKV is a neurotropic virus capable of causing congenital neurodevelopmental birth defects. As ZIKV antibodies (Abs) can potentially enhance infection by DENVs, a preventive ZIKV vaccine must be designed to eliminate antibody dependent enhancement of infection. We developed a Zika Subunit Vaccine (ZSV) consisting of two proteins, ZS and S, in a genetically pre-determined ratio of 1:4, using the methylotrophic yeast Pichia pastoris. ZS is an in-frame fusion of ZIKV envelope domain III with the Hepatitis B virus (HBV) surface antigen, and S is the un-fused HBV surface antigen. Using specific monoclonal Abs we showed the presence of ZS and S in the co-purified material which were found to co-assemble into virus-like particles (VLPs), based on dynamic light scattering and electron microscopic analyses. These VLPs were immunogenic in BALB/c mice, eliciting Abs capable of neutralizing ZIKV reporter virus particles. Further, the VLP-induced Abs did not enhance a sub-lethal DENV-2 challenge in AG129 mice. This important safety feature, coupled to the well-documented advantage of P. pastoris expression system, warrants further exploration of ZSV VLP as a possible vaccine candidate.

Assessment of the impact of manufacturing changes on the physicochemical properties of the recombinant vaccine carrier ExoProtein A.

Efforts to develop a vaccine for the elimination of malaria include the use of carrier proteins to assemble monomeric antigens into nanoparticles to maximize immunogenicity. Recombinant ExoProtein A (EPA) is a detoxified form of Pseudomonas aeruginosa Exotoxin A which has been used as a carrier in the conjugate vaccine field. A pilot-scale process developed for purification of EPA yielded product that consistently approached a preset upper limit for host cell protein (HCP) content per human dose. To minimize the risk of bulk material exceeding the specification, the purification process was redeveloped using mixed-mode chromatography resins. Purified EPA derived from the primary and redeveloped processes were comparable following full biochemical and biophysical characterization. However, using a process specific immunoassay, the HCP content was shown to decrease from a range of 0.14-0.24% w/w of total protein to below the level of detection with the revised process. The improved process reproducibly yields EPA with highly similar quality characteristics as the original process but with an improved profile for the HCP content.

Bivalent vaccine platform based on ca influenza virus vaccine elicits protective immunity against human adenoviruses.

Human adenoviruses (HAdVs) are prevalent in pediatric and adult patients with severe acute respiratory disease (ARD). To date, there have been no widely used HAdV vaccines available. In this report, we developed a cold-adapted attenuated influenza virus, termed rg HAdV-Flu ca, carrying epitopes from HAdV hexon protein in the backbone of the ca influenza vaccine neuraminidase (NA) gene using reverse genetics. Rg HAdV-Flu ca virus exhibited a cold-adapted (ca) phenotype, and its morphological characteristics were observed using electron microscopy. Moreover, BALB/c mice were immunized intranasally (i.n.) with 105, 106 or 107 TCID50 rg HAdV-Flu ca. Results showed a specific, robust antibody response against influenza and HAdV in a dose-dependent manner. More importantly, potent humoral, mucosal and cellular immune responses protected against subsequent wild-type HAdV-3 or HAdV-7 challenges, as determined by a significant decrease in viral titers and a noticeable alleviation of histopathological alterations in the lung tissue of challenged mice. These findings demonstrate that rg HAdV-Flu ca warrants attention as a potential vaccine candidate against HAdV infection.

A novel chimeric Hepatitis B virus S/preS1 antigen produced in mammalian and plant cells elicits stronger humoral and cellular immune response than the standard vaccine-constituent, S protein.

Chronic Hepatitis B Virus (HBV) infection leads to severe liver pathogenesis associated with significant morbidity and mortality. As no curable medication is yet available, vaccination remains the most cost-effective approach to limit HBV spreading and control the infection. Although safe and efficient, the standard vaccine based on production of the small (S) envelope protein in yeast fails to elicit an effective immune response in about 10% of vaccinated individuals, which are at risk of infection. One strategy to address this issue is the development of more immunogenic antigens. Here we describe a novel HBV antigen obtained by combining relevant immunogenic determinants of S and large (L) envelope proteins. Our approach was based on the insertion of residues 21-47 of the preS1 domain of the L protein (nomenclature according to genotype D), involved in virus attachment to hepatocytes, within the external antigenic loop of S. The resulting S/preS121-47 chimera was successfully produced in HEK293T and Nicotiana benthamiana plants, as a more economical recombinant protein production platform. Comparative biochemical, functional and electron microscopy analysis indicated assembly of the novel antigen into subviral particles in mammalian and plant cells. Importantly, these particles preserve both S- and preS1-specific epitopes and elicit significantly stronger humoral and cellular immune responses than the S protein, in both expression systems used. Our data promote this antigen as a promising vaccine candidate to overcome poor responsiveness to the conventional, S protein-based, HBV vaccine.

Immunoprotection induced by CpG-ODN/Poly(I:C) combined with recombinant gp90 protein in chickens against reticuloendotheliosis virus infection.

The present study is focused on investigating the immunoprotective effects of CpG-ODN/Poly(I:C) combined with the viral glycoprotein gp90 protein against reticuloendotheliosis virus (REV) infection in chickens. REV's gp90 gene was amplified from the REV-infected cells and expressed in Escherichia coli (E.coli). The expressed products, upon purification, were inoculated into 7-day-old chickens with PBS, CpG-ODN or Poly(I:C) adjuvant; Two booster inoculations were then conducted, and then each chicken was challenged. The presence of REV-antibodies in serum was determined weekly after the first vaccination. The viremia and immunosuppressive effects of REV infection were also monitored after the challenge. The neutralizing effects of the antisera were tested in vitro. The results showed that the recombinant gene containing REV gp90 gene was expressed into the recombinant protein with a size of 51 Kilo Dalton (KD), which could be recognized by a monoclonal antibody (MAb) against the gp90 protein. The viremia and immunosuppressive effects of avian influenza virus (AIV) vaccine caused by REV challenge in CpG-ODN group and in Poly(I:C) group were dramatically decreased. REV antibody with low titers was induced in gp90 group and the inoculated chickens were partly protected. Compared with those in gp90 group, the titers and the positive ratios of REV antibody in CpG+gp90 group were significantly increased, whereas the viremia and immunosuppressive effects of AIV vaccine caused by REV infection were significantly decreased. In the Poly(I:C) +gp90 group, the viremia and immunosuppressive effects caused by REV infection were also dramatically decreased, although REV antibody responses were softly increased. The diluted antisera from the vaccinated chickens in both groups could completely inhibit the replication of REV in chick fibroblast cells (CEF). Hence, it can be concluded that CpG-ODN or the Poly(I:C) adjuvant can enhance the antiviral effects of the REV subunit vaccine against REV infection, which may result from different mechanisms.

A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: First-in-human trial of ChAd63-KH.

BACKGROUND: Visceral leishmaniasis (VL or kala azar) is the most serious form of human leishmaniasis, responsible for over 20,000 deaths annually, and post kala azar dermal leishmaniasis (PKDL) is a stigmatizing skin condition that often occurs in patients after successful treatment for VL. Lack of effective or appropriately targeted cell mediated immunity, including CD8+ T cell responses, underlies the progression of VL and progression to PKDL, and can limit the therapeutic efficacy of anti-leishmanial drugs. Hence, in addition to the need for prophylactic vaccines against leishmaniasis, the development of therapeutic vaccines for use alone or in combined immuno-chemotherapy has been identified as an unmet clinical need. Here, we report the first clinical trial of a third-generation leishmaniasis vaccine, developed intentionally to induce Leishmania-specific CD8+ T cells. METHODS: We conducted a first-in-human dose escalation Phase I trial in 20 healthy volunteers to assess the safety, tolerability and immunogenicity of a prime-only adenoviral vaccine for human VL and PKDL. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB. Uniquely, the latter was engineered to reflect repeat domain polymorphisms and arrangements identified from clinical isolates. We monitored innate immune responses by whole blood RNA-Seq and antigen specific CD8+ T cell responses by IFNγ ELISPOT and intracellular flow cytometry. FINDINGS: ChAd63-KH was safe at intramuscular doses of 1x1010 and 7.5x1010 vp. Whole blood transcriptomic profiling indicated that ChAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Broad and quantitatively robust CD8+ T cell responses were induced by vaccination in 100% (20/20) of vaccinated subjects. CONCLUSION: The results of this study support the further development of ChAd63-KH as a novel third generation vaccine for VL and PKDL. TRIAL REGISTRATION: This clinical trial (LEISH1) was registered at EudraCT (2012-005596-14) and ISRCTN (07766359).

Vaccine development for respiratory syncytial virus.

Respiratory syncytial virus (RSV) is an important and ubiquitous respiratory pathogen for which no vaccine is available notwithstanding more than 50 years of effort. It causes the most severe disease at the extremes of age and in settings of immunodeficiency. Although RSV is susceptible to neutralizing antibody, it has evolved multiple mechanisms of immune evasion allowing it to repeatedly infect people despite relatively little genetic diversity. Recent breakthroughs in determining the structure and antigenic content of the fusion (F) glycoprotein in its metastable untriggered prefusion form (pre-F) and the stable rearranged postfusion form (post-F) have yielded vaccine strategies that can induce potent neutralizing antibody responses and effectively boost pre-existing neutralizing activity. In parallel, novel live-attenuated and chimeric virus vaccine candidates and other novel approaches to deliver vaccine antigens have been developed. These events and activities have aroused optimism and a robust pipeline of potential vaccine products that promise to provide a means to reduce the public health burden of RSV infection.

Vaccination strategies against Zika virus.

The epidemic emergence of Zika virus (ZIKV) in 2015-2016 has been associated with congenital malformations and neurological sequela. Current efforts to develop a ZIKV vaccine build on technologies that successfully reduced infection or disease burden against closely related flaviviruses or other RNA viruses. Subunit-based (DNA plasmid and modified mRNA), viral vectored (adeno- and measles viruses) and inactivated viral vaccines are already advancing to clinical trials in humans after successful mouse and non-human primate studies. Among the greatest challenges for the rapid implementation of immunogenic and protective ZIKV vaccines will be addressing the potential for exacerbating Dengue virus infection or causing Guillain-Barré syndrome through production of cross-reactive immunity targeting related viral or host proteins. Here, we review vaccine strategies under development for ZIKV and the issues surrounding their usage.

Assessment of Pfs25 expressed from multiple soluble expression platforms for use as transmission-blocking vaccine candidates.

BACKGROUND: Transmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle. Such vaccines are envisioned as complements to vaccines that target human infection, such as RTS,S as well as drug treatment, and vector control strategies. A number of conserved proteins, including Pfs25, have been identified as promising TBV targets in research or early stage development. Pfs25 is a 25 kDa protein of Plasmodium falciparum expressed on the surface of zygotes and ookinetes. Its complex tertiary structure, including numerous cysteines, has led to difficulties in the expression of a recombinant protein that is homogeneous, with proper conformation, and free of glycosylation, a phenomenon not found in native parasite machinery. METHODS: While the expression and purification of Pfs25 in various systems, has been previously independently reported, here a parallel analysis of Pfs25 is presented to inform on the biochemical features of Pfs25 and their impact on functionality. Three scalable expression systems were used to express, purify, and evaluate Pfs25 both in vitro and in vivo, including the ability of each protein to produce functional antibodies through the standard membrane feeding assay. RESULTS: Through numerous attempts, soluble, monomeric Pfs25 derived from Escherichia coli was not achieved, while Pichia pastoris presented Pfs25 as an inhomogeneous product with glycosylation. In comparison, baculovirus produced a pure, monomeric protein free of glycosylation. The glycosylation present for Pichia produced Pfs25, showed no notable decrease in the ability to elicit transmission reducing antibodies in functional evaluation, while a reduced and alkylated Pfs25 (derived from plant and used as a control) was found to have significantly decreased transmission reducing activity, emphasizing the importance of ensuring correct disulfide stabilized conformation during vaccine design and production. CONCLUSIONS: In this study, the biochemical features of Pfs25, produced from different expression systems, are described along with their impact on the ability of the protein to elicit functional antibodies. Pfs25 expressed using baculovirus and Pichia showed promise as candidates for vaccine development.

Viral vector-based influenza vaccines.

Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors.

Hepatitis C virus infection treatment: An era of game changer direct acting antivirals and novel treatment strategies.

Chronic hepatitis C virus infection and associated liver diseases represent a major health care burden all over the world. The current standard of care, i.e. peginterferon-alfa (PEG-IFNα) plus ribavirin (RBV) are associated with frequent and sometimes serious adverse effects and contraindications, which further limit their therapeutic efficacy. The approval of first and second generation HCV protease inhibitors represents a major breakthrough in the development of novel direct acting antivirals (DAAs) against different HCV genotypes and establishes a new standard of care for chronically infected HCV genotypes 1 patients. Similarly, next generation protease inhibitors and HCV RNA polymerase inhibitors have shown better pharmacokinetics and pharmacodynamics in terms of broader HCV genotypes coverage, better safety profile, fewer drug interactions and possible once daily administration than first generation direct acting antivirals. The testing of adenovirus-based vector vaccines, which escalates the innate and acquired immune responses against the most conserved regions of the HCV genome in chimpanzees and humans, may be a promising therapeutic approach against HCV infection in coming future. This review article presents up-to-date knowledge and recent developments in HCV therapeutics, insights the shortcomings of current HCV therapies and key lessons from the therapeutic potential of improved anti-HCV treatment strategies.

Engineering and expression of a human rotavirus candidate vaccine in Nicotiana benthamiana.

BACKGROUND: Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties. RESULTS: We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2. CONCLUSION: Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher overall efficacy for the existing vaccines. The production of rotavirus proteins in plants would probably result in lower manufacturing costs, making it more affordable for developing countries. Further investigation is required to evaluate the immunogenic potential of the VLPs and fusion proteins created in this study.

Combination recombinant simian or chimpanzee adenoviral vectors for vaccine development.

Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development.

Non-chromatographic preparation of a bacterially produced single-shot modular virus-like particle capsomere vaccine for avian influenza.

Highly pathogenic avian influenza (HPAI) causes significant economic loss, reduced food security and poses an ongoing pandemic threat. Poultry vaccination significantly decreases these problems and recognizes that the health of humans, animals and ecosystems are connected. Low-cost manufacture of poultry vaccine matched quickly to the ever-changing circulating strain is needed for effective vaccination. Here, we re-engineered the process to manufacture bacterially synthesized modular capsomere comprising influenza M2e, previously shown to confer complete protection in challenged mice, for application in poultry. Modular capsomere was prepared using a simplified non-chromatographic salting-out precipitation method and its immunogenicity tested in vivo in poultry. Modular capsomere crudely purified by precipitation (pCapM2e) contained more contaminants than equivalent product purified by chromatography (cCapM2e). Unadjuvanted pCapM2e containing 80 EU of endotoxin per dose was inferior to highly purified and adjuvanted cCapM2e (2 EU per dose). However, addition of adjuvant to pCapM2e resulting in high immunogenicity after only a single dose of vaccination, yet without any local adverse reaction. This finding suggests a strong synergy between adjuvant, antigen and contaminants, and the possible existence of a "Goldilocks" level of contaminants, where high immunogenicity and low reactogenicity can be obtained in a single-shot vaccination. The simplified process offers potential cost and speed advantages to address the needs in influenza poultry vaccination in low-cost veterinary markets.

Ebola virus vaccine: benefit and risks of adenovirus-based vectors.

In 2014, an outbreak of Ebola virus spread rapidly in West Africa. The epidemic killed more than 10,000 people and resulted in transmissions outside the endemic countries. WHO hopes for effective vaccines by the end of 2015. Numerous vaccine candidates have been proposed, and several are currently being evaluated in humans. Among the vaccine candidates are vectors derived from adenovirus (Ad). Despite previous encouraging preclinical and Phase I/II trials, Ad vectors used in three Phase II trials targeting HIV were prematurely interrupted because of the lack of demonstrated efficacy. The vaccine was not only ineffective but also led to a higher rate of HIV acquisition. In this context, the authors discuss the potential benefits, risks and impact of using Ad-derived vaccines to control Ebola virus disease.

Mapping T and B cell epitopes in sperm protein 17 to support the development of an ovarian cancer vaccine.

Ovarian cancer (OC) is the seventh most common cancer in women worldwide, and the leading cause of death from gynaecological malignancy. Immunotherapeutic strategies including cancer vaccines are considered less toxic and more specific than current treatments. Sperm surface protein (Sp17) is a protein aberrantly expressed in primary as well as in metastatic lesions in >83% of ovarian cancer patients. Vaccines based on the Sp17 protein are immunogenic and protective in animal models. To map the immunogenic regions and support the development of human Sp17 peptide based vaccines, we used 6 overlapping peptides of the human Sp17 sequence adjuvanted with CpG to immunise humanised HLA-A2.1 transgenic C57BL/6 mice, and assessed immunogenicity by ELISPOT and ELISA. No CD8 T cells were found to be induced to a comprehensive panel of 10 HLA-A2.1 or H-2K(b) binding predicted epitopes. However, one of the 6 peptides, hSp17111-142, induced high levels of antibodies and IFN-γ producing T cells (but not IL-17 or IL-4) both in C57BL/6 and in C57BL/6-HLA-A2.1 transgenic mice. C57BL/6 mice immunised with CpG adjuvanted hSp17111-142 significantly prolonged the life-span of the mice bearing the ovarian carcinoma ID8 cell line. We further mapped the immuno-dominant B and T cell epitope regions within hSp17111-142 using ELISPOT and competition ELISA. Herein, we report the identification of a single immuno-dominant B cell (134-142 aa) epitope and 2 T helper 1 (Th1) cell epitopes (111-124 aa and 124-138 aa). These result together support further exploration of hSp17111-142 peptide formulations as vaccines against ovarian cancer.

Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.