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是有利的。 D-γ-谷氨酸（D-G-PGA）均聚物的肽与rPA或TT结合。用rPA-PGA或TT-PGA（10 mcg PGA/动物）皮下免疫黑猩猩。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IgG1和IgG3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位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。免疫血清的传递阻断活性与通过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.随后使用NANP（Asn-Ala-Asn-Pro）（红细胞前寄生虫阶段的环子孢子蛋白（CSP）的重复片段）作为疫苗的早期研究。合成4个和5个NANP重复序列并与BSA缀合。这些缀合物在小鼠中具有免疫原性，在IFA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。明矾吸附增强了对两种疫苗组分的抗体产生。研究了另外的CSP衍生的四肽NVDP的免疫原性。合成了以下肽：NANPNVDP2C、NVDPNANP2C和NVDP4C，并通过硫醚键以每个蛋白质分子4至20条链与溴乙酰化BSA结合。在年轻的通用小鼠中评价这些缀合物的免疫原性，并通过ELISA测量IgG抗CSP。对于所有肽，每个蛋白质分子的最佳链数为5 - 9。使用NANPNVDP序列获得最高抗体水平，但这些水平不超过仅NANP缀合物诱导的水平。然而，将NANP缀合物与NVDP或NANPNVDP缀合物组合显著增加了抗CSP水平，在IFA中具有相应的高滴度。 我们还对获得的携带来自间日疟原虫的csp基因的修饰形式的质粒进行了测序。该基因编码间日疟原虫CSP。该蛋白质的特征在于由8至11个氨基酸组成的大量重复单元，这取决于菌株。表征了与测序质粒的csp基因相关的阅读框，并将用于产生PCR引物的信息设计为扩增编码成熟间日疟原虫CSP的csp基因，而没有信号序列和羧基末端GPI锚定序列。利用这些引物，我们成功地扩增和克隆到蛋白表达质粒的修饰基因。将该质粒转化到宿主菌E. coli DH5-α作为种子库，E. coli BL21（DE3）中进行表达。用6 M尿素和镍离子亲和层析从包涵体中纯化重组CSP。重组蛋白的氨基酸序列通过分析新质粒构建体的DNA序列数据来验证。通过SDS-PAGE和Western blot对蛋白质进行鉴定。

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.