Peptide-Protein Conjugate Vaccines

炭疽杆菌。制备来自未包囊化菌株的重组PA和几种甲酰胺处理和/或明矾吸附制剂，并间隔2个月注射3次，然后在1年后再次注射到成年志愿者中。所有制剂均安全，局部和全身反应罕见且轻微。抗体测定与许可疫苗的抗体测定相比具有优势（手稿正在编写中）。 每个载体具有不同长度和密度的D-γ-谷氨酸（D-G-PGA）的均聚物的肽与BSA、rEPA、rPA或TT结合。10- 20-mer长和10 - 15摩尔PGA每摩尔蛋白质的肽是最具免疫原性的。用rPA-PGA或TT-PGA（10 mcg PGA/动物）皮下免疫黑猩猩以制备抗D-G-PGA的人源化单克隆抗体。两只黑猩猩都对疫苗的两种成分产生了抗体。使用TT缀合物获得更高的抗PGA水平。从免疫的黑猩猩产生五个D-G-PGA特异性Fab。选择两个用于进一步研究，并转化为全长人恒定区IgG 1和IgG 3单克隆抗体（mAb）。在用强毒B进行肠内孢子攻毒前18小时，对BALB/c小鼠单次给予30 mcg剂量的任一mAb。炭疽艾姆斯菌株，赋予约40 LD-50的保护。此外，在攻击后8小时或20小时给予的两种mAb提供了显著的保护。因此，这些抗-D-G-PGA单克隆抗体单独或与抗毒素单克隆抗体组合将可用于炭疽的安全有效的暴露后治疗。 恶性疟原虫研究最多的实验性疟疾疫苗是在子孢子上表达的环子孢子蛋白（CSP）及其各种形式的合成重复单元NANP。这些疫苗是安全的和轻度免疫原性的，但它们的保护是穷人和有限的持续时间，即使与佐剂。我们使用了两种方法来提供实验性疟疾疫苗： 1. 针对性、蚊寄生虫阶段，提供阻断传播的疫苗。Pfs 25是一种低分子量蛋白，本身无免疫原性，通过酰胺、腙或硫醚键结合到自身或载体蛋白上。注射到小鼠中，所有缀合物都是免疫原性的，在再注射时具有加强应答。长期研究揭示了Pfs 25自身结合的独特性质; IgG抗体水平随时间增加，在7个月左右达到峰值，并在9个月时开始下降。与其他载体缀合的Pfs 25的抗体水平在3个月后开始下降。最好的免疫原使用己二酸二酰肼作为接头。结合物吸附到明矾上进一步增加了抗体水平。免疫血清的传播阻断活性与通过ELISA测量的抗体水平相关。用Pvs 25-Pvs 25缀合物获得了类似的结果。 2. 我们遵循早期使用NANP（Asn-Ala-Asn-Pro）作为疫苗的研究，NANP是红细胞期前寄生虫环子孢子蛋白（CSP）的重复片段。从制备痢疾杆菌1型合成O-SP寡糖蛋白和B中获得以下认识。炭疽菌胶囊衍生的肽-蛋白缀合物，我们制备了4个和5个NANP重复与BSA缀合。这些缀合物在小鼠中具有免疫原性，在免疫荧光测定中诱导具有相应高滴度的加强应答。向NANP重复序列中添加CSP T细胞表位不会增强抗CSP水平或持久性。NANP的末端氨基酸的同一性是至关重要的，其中末端Asn如NANP或NPNA是最好的免疫原，末端Ala是较差的免疫原。每个载体的肽的最佳密度为约10，4和5个重复之间没有差异。明矾吸附增强了对这两种疫苗成分的抗体产生。另外一种与载体蛋白结合的CSP衍生四肽的免疫原性正在研究中。 然后，我们将两种方法，NANP和Pfs 25结合成一种疫苗，将NANP重复序列与Pfs 25-Pfs 25结合。该实验疫苗在小鼠中诱导针对其两种组分的抗体，具有在IFA中阻断传播和结合子孢子的次级生物学活性。Pfs 25-Pfs 25将其持久的抗体特性赋予NANP。pfs 25本身既不具有免疫原性，也不是携带者。NANP-Pfs-25缀合物将提供基于个体和群体的保护。

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.