Endotoxin-free gram-negative bacterium as a system for production and secretion of recombinant proteins.

Gram-negative bacteria are common and efficient protein expression systems, yet their outer membrane endotoxins can elicit undesirable toxic effects, limiting their applicability for parenteral therapeutic applications, e.g., production of vaccine components. In the bacterial genus Sphingomonas from the Alphaproteobacteria class, lipopolysaccharide (LPS) endotoxins are replaced with non-toxic glycosphingolipids (GSL), rendering it an attractive alternative for therapeutic protein production. To explore the use of sphingomonas as a safe expression system for production of proteins for therapeutic applications, in this study, Sphingobium japonicum (SJ) injected live into embryonated hen eggs proved safe and nontoxic. Multimeric viral polypeptides derived from Newcastle disease virus (NDV) designed for expression in SJ, yielded soluble proteins which were specifically recognized by antibodies raised against the whole virus. In addition, native signal peptide (SP) motifs coupled to secreted proteins in SJ identified using whole-genome computerized analysis, induced secretion of α Amylase (αAmy) and mCherry gene products. Relative to the same genes expressed without an SP, SP 104 increased the secretion of αAmy (3.7-fold) and mCherry (16.3-fold) proteins and yielded accumulation of up to 80 µg/L of the later in the culture medium. Taken together, the presented findings demonstrate the potential of this unique LPS-free gram-negative bacterial family to serve as an important tool for protein expression for both research and biotechnological purposes, including for the development of novel vaccines and as a live bacteria delivery system for protein vaccines. KEY POINTS: • Novel molecular tools for protein expression in non-model bacteria. • Bacteria with GSL instead of LPS as a potential vector for protein delivery.

Newcastle disease virus: is an updated attenuated vaccine needed?

Newcastle disease virus (NDV) is a major cause of infectious mortality and morbidity in poultry worldwide. It is an enveloped virus with two outer-membrane proteins-haemagglutinin-neuraminidase (HN) and fusion protein (F)-that induce neutralizing antibodies. All NDV strains belong to one serotype. Yet, NDV vaccines, derived from genotype II, do not fully prevent infection or shedding of viruses from other genotypes. The aim of this study was to test if an updated vaccine is required. For this purpose, NDVs isolated from infected, albeit heavily vaccinated, flocks were genetically and immunologically characterized. Amino acid differences in F and HN protein sequences were identified between the vaccine strain and each of the isolates, some specifically at the neutralization sites. Whereas all tested isolates showed similar haemagglutination-inhibition (HI) titres, 100-100,000 times higher antibody-to-virus ratios were needed to neutralize viral propagation in embryos by the field isolates versus the vaccine strain. As a result, a model and an equation were developed to explain the phenomenon of escape in one-serotype viruses and to calculate the HI values needed for protection, depending on variation rate at key positions. In conclusion, to confer full protection against NDVs that differ from the vaccine strain at the neutralizing epitopes, very high levels of antibodies should be raised and maintained to compensate for the reduction in the number of effective epitopes; alternatively, an adjusted attenuated vaccine should be developed-a task made possible in the current era of reverse vaccinology.

Pair-epitopes vaccination: enabling offspring vaccination in the presence of maternal antibodies.

Maternally derived antibodies (MDAs) are critical for offspring protection during the first days of life. However, due to their short half-life (3-5 days), MDA levels decline rapidly and by 8-15 days post-hatch drop to below protective levels. In addition, MDAs against a specific pathogen often impede the efficient protective immune response to that pathogen by the new-born following vaccination. The combination of these two phenomena generates a gap in protection in the period between loss of MDA protection and development of vaccine-induced protective antibodies. Herein, a concept is presented that might enable effective vaccination of 1-day-old progeny in the presence of MDAs. The idea is to vaccinate mothers and their progeny with different neutralizing epitopes of the same pathogen. This will allow an effective immune response of the progeny towards neutralizing epitopes while retaining MDA protection until high levels of self-antibodies are produced. This concept is valid for various avian viruses that express two neutralizing proteins or have numerous neutralizing epitopes on the same protein, for example, Newcastle disease virus and infectious bursal disease virus, respectively. These may be used as protein subunit vaccines or live vaccines carried by a vector. This vaccination concept may overcome the gap in protection that occurs when MDAs decrease while self-immunity is still partial, to provide continuous protection at a young age.

The effect of haptens on protein-carrier immunogenicity.

The immune response against hapten is T-cell-dependent, and so requires the uptake, processing and presentation of peptides on MHC class II molecules by antigen-presenting cells to the specific T cell. Some haptens, following conjugation to the available free amines on the surface of the carrier protein, can reduce its immunogenicity. The purpose of this study was to explore the mechanism by which this occurs. Four proteins were tested as carriers and six molecules were used as haptens. The immune response to the carrier proteins was reduced > 100-fold by some of the haptens (termed carrier immunogenicity reducing haptens--CIRH), whereas other haptens did not influence the protein immunogenicity (carrier immunogenicity non-reducing haptens--nCIRH). Conjugation of the protein to a CIRH affected protein degradation by lysosomal cathepsins, leading to the generation of peptides that differ in length and sequence from those derived from the same native protein or that protein modified with nCIRH. Injection of CIRH-conjugated protein into mice induced an increase in the population of regulatory T cells. The results of this study provide a putative mechanism of action for the reduction of immune response to haptenated proteins.

Modulating the innate immune activity in murine tumor microenvironment by a combination of inducer molecules attached to microparticles.

Targeted cancer immunotherapy is challenging due to the cellular diversity and imposed immune tolerance in the tumor microenvironment (TME). A promising route to overcome those drawbacks may be by activating innate immune cells (IIC) in the TME, toward tumor destruction. Studies have shown the ability to "re-educate" pro-tumor-activated IIC toward antitumor responses. The current research aims to stimulate such activation using a combination of innate activators loaded onto microparticles (MP). Four inducers of Toll-like receptors 4 and 7, complement C5a receptor (C5aR) and gamma Fc receptor and their combinations were loaded on MP, and their influence on immune cell activation evaluated. MP stimulation of immune cell activation was tested in vitro and in vivo using a subcutaneous B16-F10 melanoma model induced in C57BL6 mice. Exposure to the TLR4 ligand lipopolysaccharide (LPS) bound to MP-induced acute inflammatory cytokine and chemokine activity in vitro and in vivo, with the elevation of CD45(+) leukocytes in particular GR-1(+) neutrophils and F4/80 macrophages in the TME. Nevertheless, LPS alone on MP was insufficient to significantly delay tumor progression. LPS combined with the C5aR ligand C5a-pep on the same MP resulted in a similar inflammation activation pattern. However, interleukin-10 levels were lower, and tumor growth was significantly delayed. Mixtures of these two ligands on separate MP did not yield the same cytokine activation pattern, demonstrating the importance of the cells' dual activation. The results suggest that combining inducers of distinct innate immune activation pathways holds promise for successful redirection of TME-residing IIC toward anti-tumoral activation.

Development of toxin-antitoxin self-destructive bacteria, aimed for salmonella vaccination.

The most common source of foodborne Salmonella infection in humans is poultry eggs and meat, such that prevention of human infection is mostly achieved by vaccination of farm animals. While inactivated and attenuated vaccines are available, both present drawbacks. This study aimed to develop a novel vaccination strategy, which combines the effectiveness of live-attenuated and safety of inactivated vaccines by construction of inducible self-destructing bacteria utilizing toxin-antitoxin (TA) systems. Hok-Sok and CeaB-CeiB toxin-antitoxin systems were coupled with three induction systems aimed for activating cell killing upon lack of arabinose, anaerobic conditions or low concentration of metallic di-cations. The constructs were transformed into a pathogenic Salmonella enterica serovar Enteritidis strain and bacteria elimination was evaluated in vitro under specific activating conditions and in vivo following administration to chickens. Four constructs induced bacterial killing under the specified conditions, both in growth media and within macrophages. Cloacal swabs of all chicks orally administered transformed bacteria had no detectable levels of bacteria within 9 days of inoculation. By day ten, no bacteria were identified in the spleen and liver of most birds. Antibody immune response was raised toward TA carrying Salmonella which resembled response toward the wildtype bacteria. The constructs described in this study led to self-destruction of virulent Salmonella enteritidis both in vitro and in inoculated animals within a period which is sufficient for the induction of a protective immune response. This system may serve as a safe and effective live vaccine platform against Salmonella as well as other pathogenic bacteria.

Protection against avian coronavirus conferred by oral vaccination with live bacteria secreting LTB-fused viral proteins.

The devastating impact of infectious bronchitis (IB) triggered by the IB virus (IBV), on poultry farms is generally curbed by livestock vaccination with live attenuated or inactivated vaccines. Yet, this approach is challenged by continuously emerging variants and by time limitations of vaccine preparation techniques. This work describes the design and evaluation of an anti-IBV vaccine comprised of E. coli expressing and secreting viral spike 1 subunit (S1) and nucleocapsid N-terminus and C-terminus polypeptides fused to heat-labile enterotoxin B (LTB) (LS1, LNN, LNC, respectively). Following chicken oral vaccination, anti-IBV IgY levels and cellular-mediated immunity as well as protection against virulent IBV challenge, were evaluated 14 days following the booster dose. Oral vaccination induced IgY levels that exceeded those measured following vaccination with each component separately. Following exposure to inactivated IBV, splenocytes isolated from chicks orally vaccinated with LNN or LNC -expressing bacteria, showed a higher percentage of CD8+ cells as compared to splenocytes isolated from chicks vaccinated with wild type or LTB-secreting E. coli and to chicks subcutaneously vaccinated. Significant reduction in viral load and percent of shedders in the vaccinated chicks was evident starting 3 days following challenge with 107.5 EID50/ml virulent IBV. Taken together, orally delivered LTB-fused IBV polypeptide-expressing bacteria induced virus-specific IgY antibody production and was associated with significantly shorter viral shedding on challenge with a live IBV. The proposed vaccine design and delivery route promise an effective and rapidly adaptable means of protecting poultry farms from devastating IB outbreaks.

Oral subunit SARS-CoV-2 vaccine induces systemic neutralizing IgG, IgA and cellular immune responses and can boost neutralizing antibody responses primed by an injected vaccine.

The rapid spread of the COVID-19 pandemic, with its devastating medical and economic impacts, triggered an unprecedented race toward development of effective vaccines. The commercialized vaccines are parenterally administered, which poses logistic challenges, while adequate protection at the mucosal sites of virus entry is questionable. Furthermore, essentially all vaccine candidates target the viral spike (S) protein, a surface protein that undergoes significant antigenic drift. This work aimed to develop an oral multi-antigen SARS-CoV-2 vaccine comprised of the receptor binding domain (RBD) of the viral S protein, two domains of the viral nucleocapsid protein (N), and heat-labile enterotoxin B (LTB), a potent mucosal adjuvant. The humoral, mucosal and cell-mediated immune responses of both a three-dose vaccination schedule and a heterologous subcutaneous prime and oral booster regimen were assessed in mice and rats, respectively. Mice receiving the oral vaccine compared to control mice showed significantly enhanced post-dose-3 virus-neutralizing antibody, anti-S IgG and IgA production and N-protein-stimulated IFN-γ and IL-2 secretion by T cells. When administered as a booster to rats following parenteral priming with the viral S1 protein, the oral vaccine elicited markedly higher neutralizing antibody titres than did oral placebo booster. A single oral booster following two subcutaneous priming doses elicited serum IgG and mucosal IgA levels similar to those raised by three subcutaneous doses. In conclusion, the oral LTB-adjuvanted multi-epitope SARS-CoV-2 vaccine triggered versatile humoral, cellular and mucosal immune responses, which are likely to provide protection, while also minimizing technical hurdles presently limiting global vaccination, whether by priming or booster programs.

Transcutaneous immunization with hydrophilic recombinant gp100 protein induces antigen-specific cellular immune response.

The objective of this study was to evaluate the potential of transcutaneous immunization with tumor antigen to induce cell-mediated immunity. For this purpose, hydrophilic recombinant gp100 protein (HR-gp100) was topically applied on human intact skin in vitro, and used as a vaccine in a mouse model. We demonstrate that HR-gp100 permeates into human skin, and is processed and presented by human dendritic cells. In a mouse model, an HR-gp100-based vaccine triggered antigen-specific T cell responses, as shown by proliferation assays, ELISA and intracellular staining for IFN-γ. Transcutaneous antigen delivery may provide a safe, simple and effective method to elicit cell-mediated immunity.

Genetic and antigenic characterization of sigma C protein from avian reovirus.

Avian reovirus (ARV) causes viral arthritis, tenosynovitis, liver infection and immunosuppression in birds. Live-attenuated and inactivated vaccines for ARV are available, but do not efficiently protect against recent variants. Sigma C, which mediates virus attachment to target cells, is the most variable protein in ARV. Antibodies to this protein neutralize viral infection. The purpose of the present study was to characterize sigma C in isolates of ARV from infected birds, as compared with the vaccine strain. Amino acids 27 to 293 of sigma C from 28 Israeli isolates were compared, classified and analysed using bioinformatics tools. Large variations were found among the isolates, and the vaccine strain was shown to differ from most of the studied strains, which could explain the failure of commonly used vaccinations in protecting birds against ARV infection. Based on sigma C protein sequences from all over the world, ARV can be divided into four groups. Isolates from all groups were found in the field simultaneously, possibly explaining the insufficient protection achieved by the vaccine strain, which is represented in one of the groups. The results point out the need and the difficulty in producing a wide-ranging vaccine. Several conserved regions among all reported ARV sigma C proteins were identified. These peptides were further studied for structural and functional properties, and for antigenic characterization. The results of this study shed light on peptide selection for a broad and efficient vaccine.

Pustulan Activates Chicken Bone Marrow-Derived Dendritic Cells In Vitro and Promotes Ex Vivo CD4+ T Cell Recall Response to Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) is a highly contagious avian coronavirus. IBV causes substantial worldwide economic losses in the poultry industry. Vaccination with live-attenuated viral vaccines, therefore, are of critical importance. Live-attenuated viral vaccines, however, exhibit the potential for reversion to virulence and recombination with virulent field strains. Therefore, alternatives such as subunit vaccines are needed together with the identification of suitable adjuvants, as subunit vaccines are less immunogenic than live-attenuated vaccines. Several glycan-based adjuvants directly targeting mammalian C-type lectin receptors were assessed in vitro using chicken bone marrow-derived dendritic cells (BM-DCs). The ß-1-6-glucan, pustulan, induced an up-regulation of MHC class II (MHCII) cell surface expression, potentiated a strong proinflammatory cytokine response, and increased endocytosis in a cation-dependent manner. Ex vivo co-culture of peripheral blood monocytes from IBV-immunised chickens, and BM-DCs pulsed with pustulan-adjuvanted recombinant IBV N protein (rN), induced a strong recall response. Pustulan-adjuvanted rN induced a significantly higher CD4+ blast percentage compared to either rN, pustulan or media. However, the CD8+ and TCRγδ+ blast percentage were significantly lower with pustulan-adjuvanted rN compared to pustulan or media. Thus, pustulan enhanced the efficacy of MHCII antigen presentation, but apparently not the cross-presentation on MHCI. In conclusion, we found an immunopotentiating effect of pustulan in vitro using chicken BM-DCs. Thus, future in vivo studies might show pustulan as a promising glycan-based adjuvant for use in the poultry industry to contain the spread of coronaviridiae as well as of other avian viral pathogens.

Cynomolgus Monkeys (Macaca fascicularis) as an Experimental Infection Model for Human Group A Rotavirus.

Group A rotaviruses (RVA) are one of the most common causes of severe acute gastroenteritis in infants worldwide. Rotaviruses spread from person to person, mainly by faecal⁻oral transmission. Almost all unvaccinated children may become infected with RVA in the first two years of life. The establishment of an experimental monkey model with RVA is important to evaluate new therapeutic approaches. In this study, we demonstrated viral shedding and viraemia in juvenile⁻adult Macaca fascicularis orally inoculated with Wa RVA prototype. Nine monkeys were inoculated orally: seven animals with human RVA and two control animals with saline solution. During the study, the monkeys were clinically monitored, and faeces and blood samples were tested for RVA infection. In general, the inoculated animals developed an oligosymptomatic infection pattern. The main clinical symptoms observed were diarrhoea in two monkeys for three days, associated with a reduction in plasmatic potassium content. Viral RNA was detected in seven faecal and five sera samples from inoculated animals, suggesting virus replication. Cynomolgus monkeys are susceptible hosts for human Wa RVA infection. When inoculated orally, they presented self-limited diarrhoea associated with presence of RVA infectious particles in faeces. Thus, cynomolgus monkeys may be useful as animal models to evaluate the efficacy of new antiviral approaches.

A melanoma multiepitope polypeptide induces specific CD8+ T-cell response.

Strategies using epitope-based vaccination are being considered for melanoma immunotherapy, in an attempt to overcome failure of other modalities. In the present study, we designed and produced a multiepitope polypeptide for melanoma (MEP-mel), which contains three repeats of four antigenic epitopes (gp100: 209-217 (210M); gp100: 280-288 (288V); Mart1: 26-35 (27L); tyrosinase: 368-376 (370D). The peptides were attached to each other by linkers containing sequences recognized by the proteasome, to improve protein cleavage and antigen presentation. The results show that peptide-specific T cells produced IFN-gamma when stimulated with MEP-mel-transfected dendritic cells. The presentation of peptides by MEP-mel-transfected dendritic cells was proteasome-dependent and was more long-lasting than the presentation of exogenously delivered native peptides. When dendritic cells were loaded with MEP-mel protein, weak cross presentation was induced. The production of multiepitope molecules based on several peptides linked by sequences sensitive to proteasomal cleavage represents a promising new tool for the improvement of cancer immunotherapy.

Melanoma antigens and related immunological markers.

Melanoma is a highly lethal cancer deriving from transformed dermal melanocytes. Early diagnosed primary melanoma may be curable, but the cure-rate of more advanced stages is limited, with high mortality rate. With the progression of the tumor, the melanocytes overexpress intracellular or cell-surface molecules, including ectopic normal and tumor-specific proteins. Some of these induce a specific immune response by T and B lymphocytes. Antibodies raised against melanoma antigens were proposed for differential disease diagnosis, staging, prognosis and evaluation of treatment efficiency. Nevertheless, treatments based on stimulation of specific anti-melanoma immune responses have had only limited success. It seems that efficient immunotherapy should become more feasible pending on finding new adequate antigens to target. New insights into immune regulation of the tumor microenvironment and its progression may help the development of more successful treatments. We present here up-to-date information on known major melanoma-associated antigens, which could serve as tools for diagnosis as well as for clinical immunotherapy. This approach with promising results for treating some other selected malignancies is still experimental with a very limited success in melanoma. The development of new immune modulators of the tumor microenvironment and neo-antigens may be additional promising directions and may open new opportunities for the immunotherapy of melanoma.

Immune Modulation and Treatment of Human Papilloma Virus-Related Warts with Energetics of Living Systems Acupuncture.

Background: Cutaneous warts are small skin lesions formed as a result of infection by the human papilloma virus (HPV). In the lesion, viral manipulation creates a microenvironment that favors virus survival and reproduction. Most lesions eventually regress, probably as a result of a Th1-mediated immune response. However, some warts fail to regress and become persistent. Objective: The efficacy of treatment of persistent HPV-caused warts with Energetics of Living Systems acupuncture and monitored immune system involvement was tested. Methods: Eighteen patients with persistent warts were recruited for the study; 9 received acupuncture treatment and 9 received placebo. Each patient was treated 4 times. Results: Clinical success was defined as total clearance of all lesions with no recurrence for 3 months. In the treatment group, clinical success was 36.6% versus 0% in the placebo group. In the treatment group, the level of interleukin (IL)-10 decreased. In a comparison of patients with cleared warts and overall patients with nonresponding warts, different expression levels of IL-8, IL-10, tumor necrosis factor-α, IL-6, and interferon-γ were found, although these differences were not always statistically significant. Trends of differences (not significant) were observed in leukocyte levels. Acupuncture eliminated persistent warts in some of the patients, along with inducing changes in immunologic parameters. Conclusions: Taking the clinical and immunologic outcomes together, clearance of persistent warts following acupuncture might be due to a shift toward a Th1 immune response, or an anti-inflammatory effect against the lesion-induced microenvironment.

Formation of multimeric antibodies for self-delivery of active monomers.

Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

Optimized polypeptide for a subunit vaccine against avian reovirus.

Avian reovirus (ARV) is a disease-causing agent. The disease is prevented by vaccination with a genotype-specific vaccine while many variants of ARV exist in the field worldwide. Production of new attenuated vaccines is a long-term process and in the case of fast-mutating viruses, an impractical one. In the era of molecular biology, vaccines may be produced by using only the relevant protein for induction of neutralizing antibodies, enabling fast adjustment to the emergence of new genetic strains. Sigma C (SC) protein of ARV is a homotrimer that facilitates host-cell attachment and induce the production and secretion of neutralizing antibodies. The aim of this study was to identify the region of SC that will elicit a protective immune response. Full-length (residues 1-326) and two partial fragments of SC (residues 122-326 and 192-326) were produced in Escherichia coli. The SC fragment of residues 122-326 include the globular head, shaft and hinge domains, while eliminating intra-capsular region. This fragment induces significantly higher levels of anti-ARV antibodies than the shorter fragment or full length SC, which neutralized embryos infection by the virulent strain to a higher extent compared with the antibodies produced in response to the whole virus vaccine. Residues 122-326 fragment is assumed to be folded correctly, exposing linear as well as conformational epitopes that are identical to those of the native protein, while possibly excluding suppressor sequences. The results of this study may serve for the development of a recombinant subunit vaccine for ARV.

Practical aspects in the use of passive immunization as an alternative to attenuated viral vaccines.

Passive immunization as a method to protect birds has been tested for many years and shown to be effective. Its advantages over active vaccination include no use of partially virulent viruses, overcoming the gap in the level of protection at young age due to interference of maternal antibodies to raise self-immune response following active vaccination and the possible immunosuppressive effect of attenuated vaccine viruses. However, a major obstacle to its implementation is its relatively high cost which is dependent, among other things, mainly on two factors: the efficacy of antibody production, and the use of specific pathogen-free (SPF) birds for antibody production to avoid the possible transfer of pathogens from commercial layers. In this study we show efficient production of immunoglobulin Y (IgY) against four different pathogens simultaneously in the same egg, and treatment of the extracted IgY with formalin to negate the need for SPF birds. Formalin, a common registered sterilization compound in vaccine production, was shown not to interfere with the Fab specific antigen binding or Fc-complement activation of the antibody. Following injection of 1-day-old broilers with antibodies against infectious bursal disease virus, protective antibody levels were acquired for the entire period of sensitivity to this pathogen (35 days). Passive vaccination with formalin-sterilized IgY against multiple antigens extracted from one commercial egg may be a cost-effective and advantageous complementary or alternative to attenuated vaccines in poultry.

Overcoming the susceptibility gap between maternal antibody disappearance and auto-antibody production.

In the first 10-14 days of a chick's life, protection is conferred by maternal antibodies. Further broiler protection is achieved by active vaccination. However, the high level of maternal antibodies interferes with the induction of an effective immune response by vaccination at a young age. As a result, there is a gap between the reduction in protective maternal antibodies and elevation of self-produced antibodies following active vaccination. The major aim of this study was to test an approach consisting of passive and active vaccination to overcome this gap and to provide continuous resistance to infectious viral diseases during the broiler's growth period. Newcastle disease virus (NDV), which is one of the world's most prevalent infectious diseases of poultry, was tested as a model. Following subcutaneous injection of 18 hemagglutination-inhibiting (HI) units of anti-NDV immunoglobulin Y per 1-day-old chick, protective log2 antibody titers above 4 could be detected to at least 17 days of age. The combination of passive immunization on day 1 of age with attenuated live vaccination on day 10 led to high protective titers throughout the entire growth period, up to 41 days of age. Moreover, the HI titers in the group of birds immunized with the combined vaccination were significantly more homogeneous than those in the group vaccinated only with live virus. Thus, full protection against NDV of all broilers in flock during their entire growth period was achieved by a vaccination regime that combines passive immunization and live vaccination.

Recombinant hydrophilic human gp100: uptake by dendritic cells and stimulation of autologous CD8+ lymphocytes from melanoma patients.

Native gp100, a glycoprotein highly expressed in the majority of melanomas, contains several immunogenic peptides that are recognized by cytotoxic lymphocytes (CTLs) in the context of major histocompatibility complex (MHC) class I molecules. The objective of this study was to evaluate the ability of dendritic cells (DCs) from melanoma patients to take up gp100 protein and stimulate specific autologous CTL. The gp100 used in this study was a recombinant molecule with diminished hydrophobicity, HR-gp100, produced in Escherichia coli bacteria and in Pichia pastoris yeast. Stimulation of CD8+ T cells from melanoma patients with HR-gp100-loaded DC was visualized by confocal microscopy using stained target cells, and was quantitatively measured by the production of IFN-gamma using an ELISPOT assay. The results showed that HR-gp100 protein, produced either in bacteria or in yeast, when loaded on DC from melanoma patients, stimulated autologous CD8+ lymphocytes. By direct visualization, these lymphocytes were found in close contact with dead melanoma cells, and to contain membrane material transferred from stained melanoma cells; in cultures containing control lymphocytes stimulated with unloaded DC, no melanoma cell killing was observed. In ELISPOT assays, increased number of IFN-gamma-producing CD8+ T lymphocytes from patients, but not from healthy controls, were measured upon stimulation with HR-gp100-loaded DC. HR-gp100 could represent a useful tool to load DC with multiple immunogenic epitopes/antigen-derived epitopes for the immunotherapy of melanoma.