Peptide-Protein Conjugate Vaccines

炭疽芽孢杆菌：从一株未被包裹的菌株和几种甲醛处理和/或明矾吸附的制剂中制备重组PA，并分别注射3次，间隔2个月，然后在1年后再次注射给成年志愿者。所有制剂都是安全的，局部和全身反应很少，而且很轻微。抗体检测结果与获得许可的疫苗检测结果进行了比较，结果令人满意。 D-γ-谷氨酸均聚体(D-G-PGA)的多肽与RPA或TT结合。用RPA-PGA或TT-PGA(10mgPGA/只)皮下注射免疫黑猩猩。两只黑猩猩都对这两种疫苗成分产生了抗体。TT结合物可获得较高的抗PGA水平。从免疫的黑猩猩中产生了5种D-G-PGA特异的Fb。将其中两株转化为全长人恒定区IgG1和IgG3单抗。在BALB/c小鼠用炭疽杆菌强毒株气管内攻击孢子前18小时，给BALB/c小鼠单次注射30微克的单抗，可获得约40LD50的保护作用。此外，攻击后8h和20h给药的mAb均具有显著的保护作用。因此，这些抗D-G-PGA单抗或与抗毒素单抗联合使用将是一种安全有效的炭疽暴露后治疗方法。 恶性疟原虫：研究最多的实验性疟疾疫苗是表达在子孢子上的环子孢子蛋白(CSP)及其合成重复单位NANP的各种形式。这些疫苗是安全的，具有温和的免疫原性，但即使在使用佐剂的情况下，它们的保护作用也很差，而且有效期有限。我们使用了两种方法来提供实验性疟疾疫苗： 1.针对有性、蚊虫寄生期，提供传播阻断疫苗。Pfs25是一种本身不具有免疫原性的小分子蛋白质，它通过酰胺、肼或硫醚键与自身或载体蛋白结合。注射到小鼠体内，所有的结合物都是免疫原性的，再次注射时具有增强反应。长期研究发现Pfs25自身具有一种独特的结合特性；抗体水平随着时间的推移而升高，在7个月左右达到峰值，9个月开始下降，与其他载体结合的抗体水平在3个月后开始下降。以己二酸二肼为连接物的免疫原效果最好。偶联物在明矾上的吸附进一步增加了抗体水平。免疫血清的传播阻断活性与ELISA检测的抗体水平相关。用Pvs25-Pvs25结合物也得到了类似的结果。 2.用红细胞前期寄生虫环子孢子蛋白(CSP)的重复片段NANP(ASN-ALA-ASN-Pro)作为疫苗进行早期研究。合成了4个和5个NANP重复序列并与BSA偶联。这些结合物在小鼠体内具有免疫原性，在IFA中诱导了相应的高滴度的增强反应。在NANP重复序列中添加CSP T细胞表位并没有提高抗CSP水平或持久性。NANP末端氨基酸的一致性是关键，末端ASN是NANP或NPNA，是最好的免疫原，末端ALA是较差的免疫原。每个载体的最佳多肽浓度为10左右，重复4次和5次之间没有差异。明胶吸附增强了对这两种疫苗成分的抗体产生。对CSP衍生的四肽NVDP的免疫原性进行了研究。合成了NANPNVDP2C、NVDPNANP2C和NVDP4C三种多肽，并通过硫醚键与溴乙酰化牛血清白蛋白结合，每个蛋白质分子有4~20条链。用EL ISA法检测其免疫原性和免疫原性。对于所有的多肽，每个蛋白质分子的最佳链数在5-9个之间。使用NANPNVDP序列获得的抗体水平最高，但这些水平没有超过仅使用NANP结合物诱导的水平。然而，将NANP结合物与NVDP或NANPNVDP结合物结合，可显著增加IFA中相应高滴度的抗CSP水平。 我们还对从间日疟原虫中获得的携带CSP基因修饰形式的质粒进行了测序。该基因编码间日疟原虫CSP。这种蛋白质的特征是根据菌株的不同，由8到11个氨基酸组成的大量重复单位。鉴定了与测序质粒的CSP基因相关的阅读框架，以及用于产生用于扩增编码成熟间日疟原虫CSP基因的PCR引物的信息，该序列不包括信号序列和羧基末端的GPI-锚序列。利用这些引物，我们成功地扩增出了修饰基因，并将其克隆到蛋白表达载体中。转化宿主菌DH5-α作为种源，转化大肠杆菌BL21(DE3)进行表达。用6M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经DNA序列分析得到证实。用SDS-PAGE和Western印迹对该蛋白进行了进一步鉴定。

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.