Bacillus anthracis: A recombinant PA from an uncapsulated strain and several formaldehyde-treated and/or alum-adsorbed formulations were prepared and injected 3 times, 2 months apart, followed by another injection 1 year later into adult volunteers. All formulations were safe and local and systemic reactions were rare and minor. Antibody assays compared favorably with those of the licensed vaccine. Peptides of the homopolymer of D-gamma-glutamic acid (D-G-PGA) were bound to rPA or TT. Chimpanzees were immunized sc with rPA-PGA or TT-PGA (10 mcg PGA/animal). Both chimps responded with antibodies to both vaccine components. Higher anti-PGA levels were obtained with the TT conjugate. Five D-G-PGA-specific Fabs were generated from immunized chimpanzees. Two were selected for further study and converted into full-length human constant region IgG1 and IgG3 monoclonal antibodies (mAb). A single 30 mcg dose of either mAb, given to BALB/c mice 18 h before intratracheal spore challenge with the virulent B. anthracis Ames strain, conferred protection from about 40 LD50. Also, both mAb given 8 h or 20 h after challenge provided significant protection. Thus, these anti-D-G-PGA mAbs would be useful, alone or in combination with anti-toxin mAbs, for a safe and efficacious postexposure therapy for anthrax. Plasmodium falciparum: The most studied experimental malaria vaccines have been the circumsporozoite protein (CSP), expressed on the sporozoite, and various forms of its synthesized repeat unit, NANP. These vaccines were safe and mildly immunogenic, but their protection was poor and of limited duration even when administered with adjuvants. We used two approaches to provide experimental malaria vaccines: 1. Directed to the sexual, mosquito parasite stage, to provide a transmission blocking vaccine. Pfs25, a low molecular mass protein, non immunogenic by itself, was bound onto itself or to carrier proteins by amide, hydrazone or thioether linkages. Injected into mice, all conjugates were immunogenic with booster responses upon reinjection. Long term studies revealed a unique property of Pfs25 bound onto itself;IgG antibody levels increased with time, peaking at around 7 months and starting to decline at 9. Antibody levels of Pfs25 conjugated to other carriers started to decline after 3 months. The best immunogens used adipic acid dihydrazide as the linker. Adsorption of the conjugates onto alum increased further the antibody levels. Transmission blocking activity of immune sera correlated with antibody levels measured by ELISA. Similar results were obtained with Pvs25-Pvs25 conjugates. 2. Followed earlier studies using NANP (Asn-Ala-Asn-Pro), the repeat fragment of the circumsporozoite protein (CSP) of the pre-erythrocytic parasite stage as a vaccine. Four and 5 NANP repeats were synthetized and conjugated to BSA. These conjugates were immunogenic in mice, induced booster responses with corresponding high titers in IFA. The addition of a CSP T-cell epitope to the NANP repeats did not enhance anti-CSP levels or persistence. The identity of the terminal amino acid of NANP was critical, with a terminal Asn as NANP or NPNA, being the best immunogens, a terminal Ala was a poor immunogen. The optimal density of the peptide per carrier was around 10 with no difference between 4 and 5 repeats. Alum adsorption enhanced antibody production to both vaccine components. The immunogenicity of an additional CSP-derived tetrapeptide, NVDP, was studied. The following peptides were synthesized: NANPNVDP2C, NVDPNANP2C and NVDP4C and bound to bromoacetylated BSA through thioether linkages at 4 to 20 chains per protein molecule. The immunogenicity of these conjugates was evaluated in young general purpose mice and IgG anti CSP measured by ELISA. For all peptides the optimal number of chains per protein molecule was between 5-9. The highest antibody levels were obtained using the NANPNVDP sequence but these levels did not exceed those induced by NANP only conjugates. However, combining a NANP conjugate with either NVDP or NANPNVDP conjugate increased significantly anti-CSP levels with corresponding high titers in IFA. We have also sequenced an obtained plasmid carrying a modified form of the csp gene from Plasmodium vivax. This gene codes for the P. vivax CSP. The protein is characterized by the large number of repeat units made up of 8 to 11 amino acids depending on the strain. The reading frame related to the csp gene of the sequenced plasmid was characterized, and the information used to produce PCR primers designed to amplify a csp gene encoding the mature P. vivax CSP, without the signal sequence and the carboxyl-terminal GPI-anchor sequence. Using these primers, we successfully amplified and cloned the modified gene into a protein expression plasmid. The plasmid was transformed into host E. coli DH5-alpha for seed stocks and E. coli BL21(DE3) for expression. The recombinant CSP was purified from inclusion bodies using 6 M urea and Ni-ion affinity chromatography. The amino acid sequence of the recombinant protein was verified by analysis of the DNA sequence data of the new plasmid construct. The protein was further characterized by SDS-PAGE and Western blot.
|Chen, Zhaochun; Schneerson, Rachel; Lovchik, Julie et al. (2011) Pre- and postexposure protection against virulent anthrax infection in mice by humanized monoclonal antibodies to Bacillus anthracis capsule. Proc Natl Acad Sci U S A 108:739-44|
|Kubler-Kielb, Joanna; Majadly, Fathy; Biesova, Zuzana et al. (2010) A bicomponent Plasmodium falciparum investigational vaccine composed of protein-peptide conjugates. Proc Natl Acad Sci U S A 107:1172-7|
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