Peptide-Protein Conjugate Vaccines

炭疽杆菌。从未封装的菌株和几种甲醛处理和/或铝吸附的配方中制备重组PA，间隔2个月注射3次，1年后再次注射给成年志愿者。所有的配方都是安全的，局部和全身反应是罕见的和轻微的。抗体检测结果优于已获得许可的疫苗（手稿正在准备中）。

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 (manuscript in preparation). Peptides of the homopolymer of D-gamma-glutamic acid (D-G-PGA) of various lengths and densities per carrier were bound to BSA, rEPA, rPA or TT. Peptides of 10- to 20-mer long and 10 to 15 mole PGA per mole protein were the most immunogenic. Chimpanzees were immunized sc with rPA-PGA or TT-PGA (10 mcg PGA/animal) for preparing humanized monoclonal antibodies to D-G-PGA. 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 (mAbs). 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 LD-50. 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. We followed earlier studies of using NANP (Asn-Ala-Asn-Pro), the repeat fragment of the circumsporozoite protein (CSP) of the pre-erythrocytic parasite stage as a vaccine. Following knowledge we gained from preparing shigella dysenteriae type1 synthetic O-SP oligosaccharide-protein and B. anthracis capsule-derived peptide-protein conjugates, we prepared 4 and 5 NANP repeats conjugated to BSA. These conjugates were immunogenic in mice, induced booster responses with corresponding high titers in the immunofluorescent assay. 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.Immunogenicity of an additional CSP-derived tetrapeptide bound by to a carrier protein is being studied. We then combined the two approaches, NANP and Pfs25 into one vaccine, binding NANP repeats to Pfs25-Pfs25. This experimental vaccine induced in mice antibodies to both its components, with secondary biological activities of transmission blocking and binding to sporozoites in IFA. The Pfs25-Pfs25 imparted its long lasting antibody properties to NANP. Pfs25 by itself was not immunogenic nor a carrier. The NANP-Pfs25-Pfs-25 conjugates would provide both individual and community based protection.