Use In Vaccine Trials Research Articles

Development and validation of glycoprotein-based native-subunit vaccine for fish against Aeromonas hydrophila.

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein-based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A.hydrophila isolates from fish farms, one A.hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA-specific lectin and size-exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)3 ] and Group IV [with Al(OH)3 and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A.hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.

Divergent opinions of proper Lyme disease diagnosis and implications for children co-morbid with autism spectrum disorder

This paper proposes that some children with an autism spectrum disorder (ASD) in the United States have undiagnosed Lyme disease and different testing criteria used by commercial laboratories may be producing false negative results. Two testing protocols will be evaluated; first, the Centers for Disease Control (CDC) and Infectious Disease Society of America (IDSA) approved two-tiered Enzyme Immunoassay (EIA) or Immunofluorescence Assay (IFA) followed by an IgM and/or IgG Western Blot test. Second, a clinical diagnosis (flu like symptoms, joint pain, fatigue, neurological symptoms, etc.) possibly followed by a Western Blot with a broader criteria for positive bands [1]. The hypothesis proposes that the former criteria may be producing false negative results for some individuals diagnosed with an ASD. Through an online survey parents of 48 children who have a diagnosis of an ASD and have been diagnosed with Lyme disease were asked to fill out the Autism Treatment Evaluation Checklist (ATEC) before they started antibiotic therapy and after treatment. Of the 48 parents surveyed 45 of them (94%) indicated their child initially tested negative using the two-tiered CDC/IDSA approved test. The parents sought a second physician who diagnosed their child with Lyme disease using the wider range of Western Blot bands. The children were treated with antibiotics and their scores on the ATEC improved. Anecdotal data indicated that some of the children achieved previously unattained developmental milestones after antibiotic therapy began. Protein bands OSP-A and/or OSP-B (Western Blot band 31) and (Western Blot band 34) were found in 44 of 48 patients. These two bands are so specific to Borrelia burgdorferi that they were targeted for use in vaccine trials, yet are not included in the IDSA interpretation of the Western Blot.

Placebo use in vaccine trials: Recommendations of a WHO expert panel

Vaccines are among the most cost-effective interventions against infectious diseases. Many candidate vaccines targeting neglected diseases in low- and middle-income countries are now progressing to large-scale clinical testing. However, controversy surrounds the appropriate design of vaccine trials and, in particular, the use of unvaccinated controls (with or without placebo) when an efficacious vaccine already exists. This paper specifies four situations in which placebo use may be acceptable, provided that the study question cannot be answered in an active-controlled trial design; the risks of delaying or foregoing an efficacious vaccine are mitigated; the risks of using a placebo control are justified by the social and public health value of the research; and the research is responsive to local health needs. The four situations are: (1) developing a locally affordable vaccine, (2) evaluating the local safety and efficacy of an existing vaccine, (3) testing a new vaccine when an existing vaccine is considered inappropriate for local use (e.g. based on epidemiologic or demographic factors), and (4) determining the local burden of disease.

Assessment of Goat Tuberculosis Model for Use in Vaccine Trials

Progressin development of tuberculosis (TB) vaccines largely depends on the availability of animal models to test their safety and efficacy before starting with expensive clinical trials. The present study provides a comprehensive evaluation of bacillus Calmette-Guerin (BCG) effects on clinical, immunological, pathological and bacteriological parameters in goats after an experimental challenge with Mycobacterium caprae.Vaccination of goats with BCG reduced the volume of lung gross lesions, the bacterial load in pulmonary lymph nodes and increased the weight gain when compared to unvaccinated animals. Differences in post-challenge IFN-γ responses to ESAT-6/CFP-10 were found to be a useful follow-up biomarker of disease progression and vaccine efficacy. Our results endorse this animal model for further TB vaccine trials.

Goats Primed with Mycobacterium bovis BCG and Boosted with a Recombinant Adenovirus Expressing Ag85A Show Enhanced Protection against Tuberculosis

This is the first efficacy study using the experimental goat model, a natural host of tuberculosis (TB), to evaluate the efficacy of heterologous Mycobacterium bovis bacillus Calmette-Guérin (BCG) prime followed by boosting with a replication-deficient adenovirus expressing the antigen Ag85A (AdAg85A). Three experimental groups of 11 goat kids each were used: BCG vaccinated, BCG vaccinated and AdAg85A boosted, and nonvaccinated. Twenty-two goat kids were vaccinated with ∼5 × 10(5) CFU of BCG (week 0), and 11 of them were boosted at week 8 with 10(9) PFU of AdAg85A. At week 14, all goats were challenged by the endobronchial route with ∼1.5 × 10(3) CFU of Mycobacterium caprae. The animals were euthanized at week 28. Cellular and humoral immunity induced by vaccination and M. caprae infection was measured throughout the study. After challenge BCG-AdAg85A-vaccinated animals exhibited reduced pathology compared to BCG-vaccinated animals in lungs and in pulmonary lymph nodes. There were significant reductions in bacterial load in both groups of vaccinated goats, but the reduction was more pronounced in prime-boosted animals. Antigen-specific gamma interferon (IFN-γ) and humoral responses were identified as prognostic biomarkers of vaccination outcome depending on their correlation with pathological and bacteriological results. As far as we know, this is the first report using multidetector computed tomography (MDCT) to measure vaccine efficacy against pulmonary TB in an animal model. The use in vaccine trials of animals that are natural hosts of TB may improve research into human TB vaccines.

Differential Neutralization of Human Immunodeficiency Virus (HIV) Replication in Autologous CD4 T Cells by HIV-Specific Cytotoxic T Lymphocytes

Defining the antiviral efficacy of CD8 T cells is important for immunogen design, and yet most current assays do not measure the ability of responses to neutralize infectious virus. Here we show that human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) clones and cell lines derived from infected persons and targeting diverse epitopes differ by over 1,000-fold in their ability to retard infectious virus replication in autologous CD4 T cells during a 7-day period in vitro, despite comparable activity as assessed by gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. Cell lines derived from peripheral blood mononuclear cells stimulated in vitro with peptides representing targeted Gag epitopes consistently neutralized HIV better than Env-specific lines from the same person, although ineffective inhibition of virus replication is not a universal characteristic of Env-specific responses at the clonal level. Gag-specific cell lines were of higher avidity than Env-specific lines, although avidity did not correlate with the ability of Gag- or Env-specific lines to contain HIV replication. The greatest inhibition was observed with cell lines restricted by the protective HLA alleles B*27 and B*57, but stimulation with targeted Gag epitopes resulted in greater inhibition than did stimulation with targeted Env epitopes even in non-B*27/B*57 subjects. These results assessing functional virus neutralization by HIV-specific CD8 T cells indicate that there are marked epitope- and allele-specific differences in virus neutralization by in vitro-expanded CD8 T cells, a finding not revealed by standard IFN-gamma ELISPOT assay currently in use in vaccine trials, which may be of critical importance in immunogen design and testing of candidate AIDS vaccines.

Identification of a Highly Immunogenic HLA-A*01-Binding T Cell Epitope of WT1

The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of WT1 which could be useful for immunotherapy. Potential HLA-A*01 epitopes predicted by a MHC binding algorithm were screened for recognition by peripheral blood mononuclear cells (PBMC) from patients with spontaneous T cell responses using intracellular cytokine cytometry. Epitope processing was shown by proteasomal cleavage. Epitope-specific T cells were generated from CD4+CD25+ regulatory T cell-depleted PBMC. One of five predicted HLA-A*01-binding candidate epitopes showed high immunogenicity as 5 of 14 patients with hematologic malignancies had WT1.317-327-reactive T cells ranging from 0.4% to 1.5% of CD3+CD8+ T cells. Proteasomal degradation assays indicated the cleavage of WT1.317-327. The depletion of regulatory T cells from PBMCs enabled the rapid expansion of WT1.317-327-specific CTL, whereas no CTL could be generated from unfractionated PBMC. WT1.317-327-specific CTL efficiently lysed an autologous WT1-expressing tumor cell line but not HLA-A*01-negative WT1-expressing tumor cells. Immunogenicity of the epitope across histologies was verified by the demonstration of spontaneous ex vivo WT1.317-327-specific T cell responses in two of six patients with HLA-A*01-positive melanoma or lung cancer. In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*01-restricted T cell epitope of the tumor antigen WT1, which is of considerable interest for use in vaccination trials.

Differential gene expression identifies novel markers of CD4+ and CD8+ T cell activation following stimulation by Mycobacterium tuberculosis.

T cell activation in response to antigenic stimulation is a complex process, involving changes in the expression level of a large number of genes. We have used cDNA array technology to characterize the differences in gene expression between human CD4+ and CD8+ T cells. PBMC from six healthy donors were stimulated with live Mycobacterium tuberculosis, and the gene expression profiles of each donor's CD4+ and CD8+ T cells were analyzed separately. ANOVA revealed 518 genes that were consistently differentially expressed between CD4+ and CD8+ T cells. These differentially expressed genes include a combination of well-known, previously characterized genes with a range of biological functions and unknown in silico predicted hypothetical genes. Where possible, the novel genes have been characterized using bioinformatics, and putative transcription factors, signaling molecules, transmembrane, and secreted factors have been identified. A subset of these differentially expressed genes could be exploited as markers of CD4+ and CD8+ T cell activation for use in vaccine trials. These observed differences in the gene expression profile of CD4+ and CD8+ T cells following activation by a human pathogen contribute to an increased understanding of T cell activation and differentiation and the roles these T cell subsets may play in immunity to infection.

Recombinant expression and antigenic properties of a 31.5-kDa keratinolytic subtilisin-like serine protease from Microsporum canis

A secreted 31.5-kDa keratinolytic subtilase (SUB3; AJ431180) is thought to be a Microsporum canis virulence factor and represents a candidate for vaccination trials. In this study, the recombinant keratinase (r-SUB3) was produced by the Pichia pastoris expression system and purified to homogeneity. Recombinant SUB3 displayed identical biochemical properties with the native protease. Experimentally cutaneously infected guinea pigs showed specific lymphoproliferative response towards r-SUB3, while no specific humoral immune response was induced except for one animal. The heterologous expression of SUB3 provides a valuable tool for addressing further investigations on the role of this keratinase in the specific cellular immune response and on its use in vaccination trials in the cat.

CDNA Library construction and isolation of genes for candidate vaccine antigens from Chrysomya bezziana (the Old World screwworm fly).

The construction and use of cDNA libraries for the isolation of genes encoding candidate antigens for use in a recombinant vaccine against Chrysomya bezziana is described. RNA was isolated and mRNA purified from first and third instar larvae of Chrysomya bezziana and used in the synthesis of two cDNA libraries in the bacteriophage vector λ ZAP express®. These libraries were screened using Digoxigenin-labeled DNA probes obtained from two independent approaches. First, a homolog approach used probes designed from previously characterized peritrophic membrane genes identified from the related myiasis fly, Lucilia cuprina. Secondly, a de novo approach used amino-terminal and internal peptide sequence information derived from purified Chrysomya bezziana peritrophic membrane proteins to generate DNA probes. Three peritrophic membrane genes were identified and characterized. Chrysomya bezziana peritrophin-48 was identified using the homolog approach and, Chrysomya bezziana peritrophin -15 and Chrysomya bezziana peritrophin-42 were identified using the de novo approach. The identification of these genes as encoding candidate antigens against Chrysomya bezziana has allowed the production of recombinant proteins for use in vaccination trials. Key words: cDNA library, peritrophin, peritrophic membrane , Chrysomya bezziana , Lucilia cuprina , vaccine

DNA immunization of mice against SIVmac239 Gag and Env using Rev-independent expression plasmids.

Simian immunodeficiency virus (SIV) structural gene expression, including gag and env, strictly depends on the interaction of the viral posttranscriptional regulator Rev with its target RNA, the Rev-responsive element (RRE). A small RNA element, termed the constitutive transport element (CTE), located in the 3' portion of simian retrovirus 1 (SRV-1) mRNA, can efficiently substitute for the human immunodeficiency virus (HIV) Rev-RRE interaction, and thus render HIV expression and replication Rev independent. We tested the ability of the SRV-1 CTE to drive the expression of SIVmac239 env and gag from subgenomic constructs designed for possible use in vaccine trials. In vitro expression studies showed that when the SRV-1 sequence is coupled to the SIV gag and env mRNAs, it functions in an orientation-dependent fashion, and leads to strong expression of SIV Gag and Env in human and monkey cell lines; levels of CTE-mediated protein expression were similar to those obtained with a functional Rev-RRE system. On the other hand, in murine fibroblast-like cells, SIV Gag and Env were expressed from constructs at relatively high levels even in the absence of Rev-RRE; nevertheless, their expression was increased by the presence of the SRV-1 CTE. As reported previously for HIV, the murine cell lines appeared to be defective for Rev-RRE activity, and required overexpression of Rev to induce a Rev response. Intramuscular injection of the gag-CTE and env-CTE constructs in BALB/c mice resulted in the expression of the corresponding mRNAs, and the production of anti-Gag and anti-Env antibodies, thus suggesting that these vectors might be used for genetic immunization approaches.

Identification and expression of antigens from Lepeophtheirus salmonis for use in vaccination trials.

The species hpeophtheirus salmonis is an ectoparasitic louse which specifically parastises salmonids. With the introduction of marine farming of Atlantic salmon (Salmo salar), L salmonis has become an important pathogen which occurs in large numbers and causes significant losses for the fish farming industry. The copepod feeds on the mucus, blood and skin of its hosts [ 1.21 causing irritation and lesions. Loss in production occurs directly from fish mortalities due to osmotic stress, and indirectly from probable reductions in growth, secondary infections such as vibriosis [3] or from increased vulnerability to ultraviolet radiation damage [4]. The current treatment for infestations is the administration of organophosphate insecticides which have limited efficacy against the parasites [3] while being toxic to the fish [5,6] and to the flora and fauna which surround the sea cages [7]. It is also now apparent that this parasite species is becoming resistant to the organophosphate insecticides [8]. We are currently engaged in developing a vaccine against the parasite species L salmiiis which will disrupt its life-cycle and so reduce the parasite burden on farmed fish. In this study, we report on the identification and expression of hidden antigens from L. salmonis for use in vaccination trials. Monoclonal antibodies were raised against L. salmonis using a modification of the proceedure described by [9]. Immunohistochemistry was carried out on adult female L salmonis using the peroxidase anti-peroxidase techniques described by [lo]. Monoclonal antibodies where selected on the basis of immunohistochemical staining of the gut and reproductive organs of L salmonis. Monoclonal antibodies were subsequently used to screen L salmonis genomic DNA libraries constructed in kgtll using the method of [l 11. L. salmonis genomic DNA inserts from isolated lambda clones were sub-cloned into the bacterial expression vector pMAL-c2 (New England Biolabs). Cloning and expression were carried out as per manufacturer's instructions. ..

Predicted disulfide-bonded structures for three uniquely related proteins of Plasmodium falciparum, Pfs230, [formula omitted] and Pf12

Pfs230 is a surface protein of the gametes of Plasmodium falciparum and has been demonstrated to be a target of malaria transmission-blocking antibodies; it is an important andidate candidate antigen for a transmission-blocking vaccine. The target epitopes of transmission-blocking antibodies against Pfs230 are almost all reduction sensitive suggesting that disulfide bonds are critical for folding the native molecule. Following the cloning of the Pfs230 gene attempts are now underway to express subunits of the protein for use in vaccine trials. It will be important to understand the disulfide-bond structure of the Pfs230 to achieve this goal. In this paper we present a model for this structure based on the observation that the Pfs230 molecule contains a series of regularly repeated cysteine-containing motifs. Four such motifs have been identified, together with a fifth cysteineless motif, which occur in the same relative order, with regular alternating omission of specific motifs, 14 times throughout the length of the protein. Each of the 14 sets of motifs contains an even number of cysteine residues (2, 4 or 6). We postulate that each set folds into a separate disulfide-bonded domain in which corresponding pairs of cysteines form an equivalent disulfide bond in every such domain. The postulated bonding arrangements in the different domains are mutually confirmatory throughout the sequence of Pfs230. We have identified two other malaria proteins, Pfs48 45 and Pf12, which share the same arrangements of motifs and conform to the same disulfide-bond structure proposed for Pfs230; no other proteins in the sequence data base share these characteristics. Although having similarities to the ‘cystine knot’ described in other proteins, the arrangement proposed here appears to be unique among described structures.

Preparation of recombinant glycoprotease of Pasteurella haemolytica A1 utilizing the Escherichia coli alpha-hemolysin secretion system.

Three murine monoclonal antibodies were prepared against the recombinant glycoprotease of Pasteurella haemolytica A1 expressed in Escherichia coli. These monoclonal antibodies were able to recognize the authentic glycoprotease from P. haemolytica A1 culture supernatant. A recombinant plasmid which contained most of the glycoprotease gene of P. haemolytica A1 fused with the secretion signal sequence from hlyA of the E. coli alpha-hemolysin determinant was constructed. This recombinant plasmid expressed a fusion protein (Gcp-F) which was secreted into the culture supernatant by E. coli cells when the alpha-hemolysin secretion functions HlyB and HlyD are supplied in trans. Gcp-F could be readily recovered from the supernatant free from other cellular materials and is suitable for use in vaccine trials and challenge experiments in animals.