Peptide-Protein Conjugate Vaccines

炭疽芽孢杆菌是人类致命感染的原因，也是一种生物恐怖主义武器，它有两个基本的毒力因子，没有这两个因子中的任何一个都不会对人类致病。这些因素是炭疽毒素和胶囊。该毒素由致死性因子（LF）、水肿因子（EF）和保护性抗原（PA） 3种蛋白组成，其本身均无毒。PA是与哺乳动物细胞结合的毒素部分。一个20kda的肽必须从PA水解，暴露出LF或EF可能结合的位点，产生毒素，酶修饰哺乳动物细胞质中的底物。该胶囊由聚d - γ -谷氨酸（PGA）组成。它是非免疫原性的，其保护作用尚不清楚。获得许可的疫苗是安全且具有保护作用的，但其局限性证明开发改进疫苗是合理的。从未被荚膜的菌株中分离出重组PA。几种甲醛处理和明矾吸附材料的配方在小鼠中被发现具有免疫原性。这些制剂的临床评价已经开始。该胶囊从一株无毒菌株中分离得到，其或相应的合成肽与BSA、rEPA或rPA结合。为了确定最佳结构，将长度为5、10和20米的D或L构型肽与C或N端活性基团偶联。通过理化和免疫学实验对其进行了表征，并对5 ~ 6周龄小鼠进行了免疫原性研究。与单独的PGA相比，所有缀合物均具有免疫原性，PGA诱导的抗体具有调理自噬性。rPA是最有效的载体。其他偶联方法在小鼠中产生免疫原性偶联物，诱导水平与我们的出版物中描述的没有统计学差异。rPA-PGA偶联物在0.31 ~ 20 mcg/只小鼠的剂量反应实验表明，1.25 ~ 2.5 mcg对PGA反应最优，而PA抗体水平随着免疫剂量的增加而增加。明矾佐剂的使用增加了pga抗体水平，而对抗pga水平影响不大。

BACILLUS ANTHRACIS, a cause of lethal human infection and a bioterrorist weapon, has 2 essential virulence factors without either of which it is not pathogenic for humans. These factors are the anthrax toxin, and the capsule. The toxin is composed of 3 proteins: Lethal Factor (LF), Edema Factor (EF) and Protective Antigen (PA), each by itself non toxic. PA is the toxin part that binds to mammalian cells. A 20 KDa peptide must be hydrolyzed off PA, exposing a site to which LF or EF may bind, rendering toxins that enzymatically modify substrates in the mammalian cell cytosol. The capsule is composed of poly-D-gamma-glutamic acid (PGA).It is non-immunogenic and its protective effect unknown. The licensed vaccine is safe and protective but has limitations that justify development of improved vaccines. A recombinant PA was isolated from an uncapsulated strain. Several formulations with formaldehyde treated and alum adsorbed materials were found to be immunogenic in mice. Clinical evaluation of these formulations has begun. The capsule has been isolated from a non toxic strain and it or corresponding synthetic peptides were bound to BSA, rEPA or rPA. To identify the optimal construct peptides of varying lengths: 5, 10 and 20-mers, of D or L configuration with active groups at the C or N terminus were conjugated. The conjugates were characterized by physico-chemical and immunological assays and immunogenicity in 5-6 week old mice. Opposed to PGA alone all conjugates were immunogenic and the PGA induced antibodies were opsonophagocytic. rPA was the most effective carrier. Additional conjugation methods yielded conjugates immunogenic in mice, inducing levels not statistically different from those described in our publication. Dose response experiments of an rPA-PGA conjugate, with doses between 0.31 and 20 mcg/mouse showed 1.25-2.5 mcg to be optimal for a PGA response, while PA antibody levels increased with higher immunizing doses. The use of alum adjuvant increased PA antibody levels while having little effect upon anti-PGA levels. SEROSURVEY OF ARMED FORCES PERSONNEL IMMUNIZED WITH THE LICENSED ANTHRAX VACCINE: A survey was made of IgG antibodies in 246 sera sets at the U.S. Armed Forces Repository, taken from Armed Forces Personnel immunized with the FDA-licensed Anthrax Vaccine Adsorbed (AVA). Paired sera from HIV-negative personnel before and after the 3rd, 4th, and 6th injections were assayed by ELISA using purified Protective Antigen (PA) from Bacillus anthracis. The personnel were stratified according to sex and age (18-24 years and >24 years). Serum conversion rates of >= 4-fold increase in antibody levels were: pre-post 3rd, 85.3%; pre 4th-post 4th, 67.9%; and pre 6th-post 6th, 45%. Geometric mean levels of all individuals were 59.9 mcg/mL following the 3rd injection, 157.4 mcg/mL following the 4th, and 277 mcg/mL following the 6th. The levels were similar between the males and females but the GMC was statistically significantly higher for the younger vs. the older group. These values will be used to evaluate the IgG anti-PA levels induced by our investigational B. anthracis vaccines. PLASMODIUM FALCIPARUM: Malaria is a leading cause of morbidity and mortality globally, especially in children, estimated to cause over a million childhood deaths annually. P. falciparum causes the most severe form of disease. Experimental vaccines have been described and some tested clinically but no licensed vaccine is available. The most studied is the circumsporozoite protein (CS), expressed extracellularly on the sporozoite, and various forms of its synthesized repeat unit, NANP. These vaccines were safe and immunogenic but poorly protective, even when administered with adjuvants. Based on our studies with peptides of the B. anthracis capsule, peptides of 4 repeat units of NANP were synthesized and bound to carrier proteins and their immunogenicity studied in general purpose mice without adjuvants and by a scheme relevant for humans. Preliminary results showed high levels of antibodies, with circumsporozoite neutralizing activity roughly correlated to levels measured by ELISA. In another approach to provide a transmission blocking vaccine PFS25, a low molecular weight protein, was bound to carrier proteins and injected into mice to evaluate their antibody responses.