Combined conjugate vaccines: enhanced immunogenicity with the N19 polyepitope as a carrier protein

The N19 polyepitope, consisting of a sequential string of universal human CD4(+)-T-cell epitopes, was tested as a carrier protein in a formulation of combined glycoconjugate vaccines containing the capsular polysaccharides (PSs) of Neisseria meningitidis serogroups A, C, W-135, and Y. Good antibody responses to all four polysaccharides were induced by one single immunization of mice with N19-based conjugates. Two immunizations with N19 conjugates elicited anti-MenACWY antibody titers comparable to those induced after three doses of glycoconjugates containing CRM197 as carrier protein. Compared to cross-reacting material (CRM)-based constructs, lower amounts of N19-MenACWY conjugates still induced high bactericidal titers to all four PSs. Moreover, N19-MenACWY-conjugated constructs induced faster and higher antibody avidity maturation against meningococcal C PS than CRM-based conjugates. Very importantly, N19-specific antibodies did not cross-react with the parent protein from which N19 epitopes were derived, e.g., tetanus toxoid and influenza virus hemagglutinin. Finally, T helper epitopes of the N19 carrier protein were effectively generated both in vivo (after immunization with the N19 itself) and in vitro (after restimulation of epitope-specific spleen cells). Taken together, these data show that the N19 polyepitope represents a strong and valid option for the generation of improved or new combined glycoconjugate vaccines.     

Effect of carrier priming on immunogenicity of saccharide-protein conjugate vaccines

Previous studies with saccharide-protein conjugates have demonstrated that antibody responses to the saccharide can be improved by the preexistence of carrier immunity. Here we report that prior exposure to the carrier protein can either enhance or suppress antibody response to polysaccharides administered in saccharide-protein conjugates. A dose-dependent role for carrier priming in the antisaccharide antibody response to three saccharide-protein conjugate vaccines, i.e., a Streptococcus pneumoniae type 4 polysaccharide-tetanus toxoid (TT) conjugate (PS4TT), a Neisseria meningitidis group C polysaccharide-TT conjugate (MenCTT), and a N. meningitidis group C oligosaccharide-diphtheria mutant toxin conjugate (MenCCRM), was investigated. The results showed that an increase in the antipolysaccharide antibody response could be obtained for both PS4TT and MenCTT but not for MenCCRM with low-dose carrier priming (0.025 to 0.25 microgram). However, suppression of the antipolysaccharide antibody response was observed with the PS4TT and MenCTT vaccines with high-dose (25-micrograms) carrier priming. There was no suppression effect with MenCCRM. The increase in the antipolysaccharide antibody response was shown to be restricted to the immunoglobulin G1 (IgG1) subclass, whereas suppression with high-dose carrier priming affected all antipolysaccharide subclass antibodies induced by PS4TT (IgG1, IgG2b, and IgG3) and only two of the four subclass antibodies induced by MenCTT (IgG2a and IgG2b). The increase in the antipolysaccharide antibody response was also present at the antipolysaccharide IgM antibody level but was not observed at the anti-carrier IgG antibody level.     

Capsular polysaccharide-protein conjugate vaccines of carbotype 1 Vibrio vulnificus: construction, immunogenicity, and protective efficacy in a murine model.

Vibrio vulnificus causes septicemia and wound infections in immunocompromised humans. The capsular polysaccharide of Vibrio vulnificus (VvPS) is critical for virulence. We synthesized conjugate vaccines of carbotype 1 VvPS under conditions and in formulations suitable for human use. Purified VvPS was conjugated to tetanus toxoid (TT) or to inactivated V. vulnificus cytolysin or elastase by two different schemes. All conjugates elicited elevated anticapsular immunoglobulin G (IgG) and IgM and antiprotein IgG responses in mice compared with saline placebo. The conjugates prepared through caboxyl activation of VvPS (VvPS-TTa, VvPS-cytolysin, and VvPS-elastase) were more immunogenic than the one prepared through hydroxyl activation (VvPS-TTb). The protective efficacy of conjugated and unconjugated formulations of VvPS and that of protein carriers were evaluated in a mouse septicemia model. Eighty percent of mice actively immunized with VvPS-TTa vaccine survived challenge with carbotype 1 V. vulnificus, while VvPS-cytolysin and VvPS-elastase conjugates conferred 44 and 40% protection, respectively. Control mice immunized with VvPS, cytolysin, or elastase alone, or saline only, showed 70 to 100% mortality. VvPS-TTa vaccine is nontoxic, immunogenic, and protective in mice.     

三种不同载体的C群脑膜炎球菌荚膜多糖-蛋白质结合疫苗比较研究

目的 通过3种不同载体即破伤风类毒素（TT）、白喉类毒素（DT）和重组铜绿假单胞菌外毒素A（rEPA）与C群脑膜炎球菌荚膜多糖（GCMP）结合，经免疫小鼠后比较3种结合疫苗的免疫原性。方法将GCMP以ADH作为间隔剂分别与TT、DT和rEPA等3种蛋白质载体结合形成GCMP-蛋白质结合疫苗，以GCMP和3种结合疫苗免疫NIH小鼠以研究在其体内的免疫原性。结果GCMP和结合疫苗免疫NIH小鼠后，其血清ELISA结果显示多糖疫苗和结合疫苗均可诱生免疫应答，但使用GCMP免疫小鼠后仅能产生较低水平抗GCMP的IgG抗体，而用3种GCMP-蛋白质结合疫苗免疫小鼠后其血清中产生了较GCMP免疫显著增高的抗GCMP的IgG抗体，并且3种GCMP-蛋白质结合疫苗第2次、第3次免疫与初次免疫相比，IgG抗体水平均有显著升高（P〈0．001），表明GCMP-蛋白质结合疫苗具有免疫记忆和加强应答效应，且3种GCMP-蛋白质结合疫苗在小鼠体内产生的抗GCMP水平差异有统计学意义（P〈0．01）。补体介导的血清抗体体外杀菌试验结果证明，3种GCMP-蛋白质结合疫苗免疫小鼠诱导的IgG抗体均比GCMP具有增强的体外杀菌活性。结论 经3种不同载体与GCMP结合后免疫小鼠均产生了比GCMP显著增强的免疫应答。比较研究表明，rEPA是实验中GCMP-蛋白质结合疫苗较好的备选载体。     

Effect of carrier selection on immunogenicity of protein conjugate vaccines against Plasmodium falciparum circumsporozoites.

Conjugate vaccines against the sporozoite stage of Plasmodium falciparum were synthesized by covalently coupling the recombinant protein R32 [with the one-letter amino acid code of MDP-[(NANP)15NVDP]2LR] to tetanus toxoid, cholera toxin, choleragenoid, and Pseudomonas aeruginosa toxin A. Conjugates were produced by using adipic acid dihydrazide as a spacer molecule and carbodiimide as a coupling agent. The molar ratio of R32 to carrier protein ranged from 2.5:1 to 8.4:1. These conjugates were found to be stable, nontoxic, and nonpyrogenic. When adsorbed onto Al(OH)3, all conjugates were capable of inducing anti-R32 antibody. Conjugates made with either cholera toxin or Pseudomonas aeruginosa toxin A were significantly more immunogenic than those constructed with tetanus toxoid or choleragenoid. However, the magnitude of the immune response to the R32 moiety was not governed by the antibody response to the carrier protein.     

Effect of vaccination with carrier protein on response to meningococcal C conjugate vaccines and value of different immunoassays as predictors of protection

In order to plan for the wide-scale introduction of meningococcal C conjugate (MCC) vaccine for United Kingdom children up to 18 years old, phase II trials were undertaken to investigate whether there was any interaction between MCC vaccines conjugated to tetanus toxoid (TT) or a derivative of diphtheria toxin (CRM(197)) and diphtheria-tetanus vaccines given for boosting at school entry or leaving. Children (n = 1,766) received a diphtheria-tetanus booster either 1 month before, 1 month after, or concurrently with one of three MCC vaccines conjugated to CRM(197) or TT. All of the MCC vaccines induced high antibody responses to the serogroup C polysaccharide that were indicative of protection. The immune response to the MCC-TT vaccine was reduced as a result of prior immunization with a tetanus-containing vaccine, but antibody levels were still well above the lower threshold for protection. Prior or simultaneous administration of a diphtheria-containing vaccine did not affect the response to MCC-CRM(197) vaccines. The immune responses to the carrier proteins were similar to those induced by a comparable dose of diphtheria or tetanus vaccine. The results also demonstrate that, for these conjugate vaccines in these age groups, both standard enzyme-linked immunosorbent assays and those that measure high-avidity antibodies to meningococcal C polysaccharide correlated equally well with assays that measure serum bactericidal antibodies, the established serological correlate of protection for MCC vaccines.     

Control and lot release of meningococcal group C conjugate vaccines

Meningococcal group C conjugate vaccines were first introduced to the UK in 1999. To date, the vaccines have been demonstrated to have an efficacy of approximately 90% and have since been adopted by other countries worldwide. The development of control tests used for lot release of meningococcal group C vaccines has been based on those used for Haemophilus influenzae type b conjugates, the key criteria being measurement of free saccharide and conjugate integrity by physicochemical means. In future, meningococcal group C vaccines are likely to be replaced by multivalent formulations containing different components in combination. This will present a new challenge for regulatory authorities and more extensive testing will be required to ensure vaccine safety and efficacy.     

PAMAM–Triamcinolone acetonide conjugate as a nucleus-targeting gene carrier for enhanced transfer activity

The excellent transfection efficiency and viability are essential for successful gene therapy. It suggested that when bound to its glucocorticoid receptor, glucocorticoid steroid can dilate the nuclear pore complexes and facilitated the transport of pDNA into the nucleus. In this research, the two different degrees of substitution of PAMAM–triamcinolone acetonide (PAMAM–TA) conjugates were synthesised for efficient translocation of pDNA into the nucleus. The physicochemical properties of the polyplexes were investigated by agarose gel electrophoresis, Zeta-sizer and TEM. They both could form nano-size polyplexes with pDNA. The polyplexes were very stable and showed excellent buffering capacities, facilitating endosomal escape, and no obvious difference was found between them. The TA-conjugated PAMAM-mediated transfection of luciferase and EGFP genes showed better transfer activity than native PAMAM and was comparable to the PEI 25K (polyethylenimine), and lower cytotoxicity in HEK 293 and HepG 2 cells. Even with 10% serum, their transfer activity was still high relatively. In addition, confocal microscopy examination confirmed that the enhancing mechanism for enhanced gene transfer activity of PAMAM–TA conjugate may involve the nuclear translocation of the polyplex. The low substituted degree of TA to 0.22 did not interrupt its nuclear localization potency. These findings demonstrated that the TA-grafted PAMAM dendrimer is a potential candidate as a safe and efficient gene delivery carrier for gene therapy.     

Technologies for enhanced efficacy of DNA vaccines

Despite many years of research, human DNA vaccines have yet to fulfill their early promise. Over the past 15 years, multiple generations of DNA vaccines have been developed and tested in preclinical models for prophylactic and therapeutic applications in the areas of infectious disease and cancer, but have failed in the clinic. Thus, while DNA vaccines have achieved successful licensure for veterinary applications, their poor immunogenicity in humans when compared with traditional protein-based vaccines has hindered their progress. Many strategies have been attempted to improve DNA vaccine potency including use of more efficient promoters and codon optimization, addition of traditional or genetic adjuvants, electroporation, intradermal delivery and various prime-boost strategies. This review summarizes these advances in DNA vaccine technologies and attempts to answer the question of when DNA vaccines might eventually be licensed for human use.     

Carrier-mediated enhancement of cognate T cell help: the basis for enhanced immunogenicity of meningococcal outer membrane protein polysaccharide conjugate vaccine

Haemophilus influenzae type b capsular polysaccharide (PRP) conjugate vaccines, which are thought to induce T cell-dependent antibody production, induce protective responses after a single dose in individuals under 15 months of age. However, multiple doses of these vaccines are required to induce protective antibody responses in infants, with the exception of PRP conjugated to meningococcal outer membrane proteins (OMPC), which does so after a single dose. The basis for this difference is not fully understood, although others have proposed that OMPC and porins, the major protein component of OMPC, act as adjuvants or mitogens. In this report OMPC is shown to enhance CD40 ligand-mediated, T cell-dependent antibody production in mice. This paralleled the induction by OMPC of CD86, CD80 and CD40 costimulatory molecules on human neonatal and murine B cells and of Th1 cytokines. Neither porins nor lipopolysaccharide fully reproduced the effects of OMPC. These studies indicate that OMPC acts both as carrier and adjuvant, and thereby enhances T cell-dependent antibody responses in human infants.     

Characterization of a recombinant pneumolysin and its use as a protein carrier for pneumococcal type 18C conjugate vaccines.

Pneumolysin from Streptococcus pneumoniae was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified by affinity and hydroxylapatite chromatography. The purified recombinant pneumolysin (rPL), with a molecular mass of 53 kDa, had a specific activity of 3 x 10(5) hemolytic units per mg of protein on rabbit erythrocytes and reacted identically in immunodiffusion with the antisera against native pneumolysin. The rPL was used as a protein carrier to prepare conjugate vaccine with pneumococcal type 18C polysaccharide (PS18C). The PS18C was directly coupled to rPL by reductive animation or was indirectly coupled to rPL via a spacer molecule, adipic acid dihydrazide. The conjugates were nontoxic for mice and guinea pigs at 100 micrograms per dose. The immunogenicity and protective efficacy of both conjugates were tested in mice. A single dose of either of the vaccines elicited a rise in immunoglobulin G antibody production; after two booster injections of the vaccines, statistically significant booster responses (P < 0.001) to both rPL and PS18C were produced. The sera containing the antibodies to rPL were capable of neutralizing the hemolytic activity of rPL to rabbit erythrocytes and the cytotoxicity of rPL to bovine pulmonary endothelial cells. Immunization with the conjugate vaccines conferred statistically significant protection in mice against lethal challenge with type 18C pneumococci.     

不同佐剂对随机组合多表位恶性疟疾疫苗M. RCAg-1的免疫原性及体外抑虫率影响的研究

目的 研究弗氏佐剂及3种人用佐剂[Al(OH)3、Montanide ISA720、Montanide ISA51]对重组多表位蛋白M.RCAg-1(malaria random constructed antigen-1)在BALB/c小鼠及新西兰大白兔体内产生免疫效果的影响.方法 以弗氏佐剂、Al( OH)3佐剂、Montanide ISA720以及MontanideISA51佐剂分别与M.RCAg-1蛋白进行配伍,分别免疫BALB/c小鼠及新西兰白兔;采用ELISA法检测M.RCAg-1蛋白及其所组成各个表位特异的血清IgG含量;间接荧光免疫试验分析不同配伍组免疫血清对天然疟原虫抗原的识别;酶联免疫斑点试验(ELISPOT)检测特异性鼠T淋巴细胞的活化;另借助生物传感器评价抗原与不同佐剂配伍组血清的亲和力,并用体外生长抑制试验(GIA)对恶性疟原虫的体外生长的影响进行评价.结果 不同佐剂可诱发不同水平的抗体滴度,Montanide ISA51佐剂配伍组的效果与弗氏佐剂组最为接近,并可使蛋白包含的11个表位肽能都较好诱发出特异性抗体,可有效激发以IFN-γ为主的细胞免疫应答,而且Montanide ISA51佐剂组免疫产生的抗体和M.RCAg-1蛋白质的亲和力高于其他两种佐剂;而Montanide ISA720佐剂和Al(OH)3佐剂免疫组不能诱发小鼠产生显著的IFN-γ反应(P＞0.05).Montanide ISA51配伍蛋白质免疫产生的IgG在浓度为2mg/ml时对3D7和Dd2的抑制率分别为60%和100%,Al(OH)3佐剂组免疫兔血清IgG体外抑虫率较低(10%左右),而Montanide ISA720佐剂免疫组血清不能有效抑制疟原虫生长.结论 通过比较3种人用佐剂和弗氏佐剂对BALB/c小鼠和新西兰兔的免疫辅助效能显示,Montanide ISA51佐剂能很好的辅助M.RCAg-1蛋白疫苗诱发动物体内的体液免疫和细胞免疫应答,可做为疫苗的候选佐剂之一.     

Alpha C protein as a carrier for type III capsular polysaccharide and as a protective protein in group B streptococcal vaccines

The alpha C protein, a protective surface protein of group B streptococci (GBS), is present in most non-type III GBS strains. Conjugate vaccines composed of the alpha C protein and type III capsular polysaccharide (CPS) might be protective against most GBS infections. In this study, the type III CPS was covalently coupled to full-length, nine-repeat alpha C protein (resulting in III-alpha9r conjugate vaccine) or to two-repeat alpha C protein (resulting in III-alpha2r conjugate vaccine) by reductive amination. Initial experiments with the III-alpha9r vaccine showed that it was poorly immunogenic in mice with respect to both vaccine antigens and was suboptimally efficacious in providing protection in mice against challenge with GBS. Therefore, modified vaccination protocols were used with the III-alpha2r vaccine. Female mice were immunized three times with 0.5, 5, or 20 microgram of the III-alpha2r vaccine with an aluminum hydroxide adjuvant and bred. Ninety-five percent of neonatal mice born to dams immunized with the III-alpha2r vaccine survived challenge with GBS expressing type III CPS, and 60% survived challenge with GBS expressing wild-type (nine-repeat) alpha C protein; 18 and 17%, respectively, of mice in the negative control groups survived (P, <0.0001). These protection levels did not differ significantly from those obtained with the type III CPS-tetanus toxoid conjugate vaccine and the unconjugated two-repeat alpha C protein, which protected 98 and 58% of neonates from infection with GBS expressing type III CPS or the alpha C protein, respectively. Thus, the two-repeat alpha C protein in the vaccine was immunogenic and simultaneously enhanced the immunogenicity of type III CPS. III-alpha vaccines may be alternatives to GBS polysaccharide-tetanus toxoid vaccines, eliciting additional antibodies protective against GBS infection.     

Chimeric human papilloma virus-simian/human immunodeficiency virus virus-like-particle vaccines: immunogenicity and protective efficacy in macaques

Vaccines to efficiently block or limit sexual transmission of both HIV and human papilloma virus (HPV) are urgently needed. Chimeric virus-like-particle (VLP) vaccines consisting of both multimerized HPV L1 proteins and fragments of SIV gag p27, HIV-1 tat, and HIV-1 rev proteins (HPV-SHIV VLPs) were constructed and administered to macaques both systemically and mucosally. An additional group of macaques first received a priming vaccination with DNA vaccines expressing the same SIV and HIV-1 antigens prior to chimeric HPV-SHIV VLP boosting vaccinations. Although HPV L1 antibodies were induced in all immunized macaques, weak antibody or T cell responses to the chimeric SHIV antigens were detected only in animals receiving the DNA prime/HPV-SHIV VLP boost vaccine regimen. Significant but partial protection from a virulent mucosal SHIV challenge was also detected only in the prime/boosted macaques and not in animals receiving the HPV-SHIV VLP vaccines alone, with three of five prime/boosted animals retaining some CD4+ T cells following challenge. Thus, although some immunogenicity and partial protection was observed in non-human primates receiving both DNA and chimeric HPV-SHIV VLP vaccines, significant improvements in vaccine design are required before we can confidently proceed with this approach to clinical trials.     

Safety and immunogenicity of meningococcal A and C polysaccharide conjugate vaccine in adults.

A meningococcal vaccine containing group A and C polysaccharides conjugated to CRM197 was evaluated in 50 adults. Vaccinees were entered into one of five groups: 30 adults received a single dose of either 22, 11, or 5.5 micrograms of the conjugated A-C vaccine; 10 received an approved meningococcal vaccine; and 10 received saline injections. Local and systemic reactions to vaccines were recorded, and immune responses were determined. The experimental meningococcal vaccine was well tolerated, with the most frequent reaction being pain at the injection site. Both A and C polysaccharide components of the experimental vaccine were highly immunogenic, and total antibody concentrations 1 month postvaccination were not significantly different from the mean antibody concentrations among adults given the approved meningococcal vaccine. In addition, significant rises in immunoglobulin G, A, and M antibodies to both A and C polysaccharides occurred. Antibody concentrations measured at 6 and 12 months postvaccination had declined but remained significantly higher than prevaccination concentrations. Postvaccination meningococcal group C functional antibody activity increased more than 600-fold for both the polysaccharide and the conjugate vaccines. Further studies of this conjugated meningococcal vaccine are indicated for young children and infants.     

肺炎球菌荚膜多糖和溶血素结合疫苗的制备及其免疫原性的研究

目的 研制肺炎球菌荚膜多糖（PS）与肺炎球菌溶血素（蛋白）偶联结合疫苗.方法 用PCR扩增肺炎球菌溶血素（Pn）基因,将其克隆入表达载体pET-28a质粒中,然后转化大肠杆菌JM109（DE3）宿主细胞,并以IPTG诱导进行蛋白质表达,对所得蛋白质用固相Ni-NTA树脂作纯化,再用SDS-PAGE鉴定表达的纯化蛋白（rPn）.以福尔马林脱毒rPn,去除其溶血活性,然后用氨基还原法将脱毒的rPn与PS（19F血清型）偶联结合,加入铝佐剂制备成疫苗.以该疫苗腹腔注射接种小鼠,用ELISA法检测小鼠血清中抗PS及抗Pn的体水平.结果 成功扩增和克隆了Pn基因,经诱导表达得到rPn蛋白,纯化后纯度达90%以上.PS与Pn结合物经凝胶层析柱分离,其洗脱峰较PS与Pn二峰明显前移.用该结合疫苗免疫的小鼠血清中抗PS（19F）抗体及抗Pn抗体的效价均明显高于阴性对照组（P＜0.01）.结论 用基因工程技术制备的肺炎球菌溶血素作为蛋白载体,脱毒后与肺炎球菌多糖偶联而成的结合疫苗能在小鼠体内诱导出明显的免疫应答反应.     

Impact of the conjugation method on the immunogenicity of Streptococcus pneumoniae serotype 19F polysaccharide in conjugate vaccines

7vCRM (Pfizer, Inc.) and PHiD-CV (GlaxoSmithKline Biologicals) are two pneumococcal conjugate vaccines licensed for the prevention of invasive pneumococcal disease and acute otitis media caused by the vaccine serotypes of Streptococcus pneumoniae. Neither vaccine contains serotype 19A, but both contain the closely related serotype 19F. No decrease in the incidence of serotype 19A disease has been observed following the introduction of 7vCRM, suggesting that this serotype 19F-containing vaccine provides limited cross-protection against serotype 19A. To investigate the impact that conjugation methods may have on antipolysaccharide immune responses and to determine whether this limited cross-protection is characteristic of the serotype 19F polysaccharide or rather of the 19F-CRM (cross-reacting material) conjugate, we compared naturally induced antibodies against serotypes 19F and 19A with antibodies induced after vaccination with different pneumococcal conjugate vaccines. We found that conjugation of the serotype 19F polysaccharide using reductive amination (as in 7vCRM) resulted in the formation of at least one additional epitope that is not present in the native form of the 19F polysaccharide or following 19F conjugation using a bifunctional spacer (as in the prototype vaccine 7vOMPC) or cyanylation (as in PHiD-CV). We also found that pneumococcal vaccines conjugated using cyanylation induce more opsonophagocytic antibodies against serotype 19F and a considerably higher level of cross-opsonophagocytic antibodies against serotype 19A than vaccines conjugated using reductive amination. In conclusion, these results suggest that the conjugation method can influence the functionality of the antibodies induced against the homologous serotype 19F and the cross-reactive serotype 19A of S. pneumoniae.     

A sopB deletion mutation enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines

SopB is a virulence factor of Salmonella encoded by SPI-5. Salmonella sopB deletion mutants are impaired in their ability to cause local inflammatory responses and fluid secretion into the intestinal lumen and also can enhance the immunogenicity of a vectored antigen. In this study, we evaluated the effects on immunogenicity and the efficacy of a sopB deletion mutation on two Salmonella enterica serovar Typhimurium vaccine strains with different attenuating mutations expressing a highly antigenic α-helical region of the Streptococcus pneumoniae surface protein PspA from an Asd⁺-balanced lethal plasmid. After oral administration to mice, the two pairs of strains induced high levels of serum antibodies specific for PspA as well as to Salmonella antigens. The levels of antigen-specific serum immunoglobulin G (IgG) and mucosal IgA were higher in mice immunized with sopB mutants. Enzyme-linked immunospot assay results indicated that the spleen cells from mice immunized with a sopB mutant showed higher interleukin-4 and gamma interferon secretion levels than did the mice immunized with the isogenic sopB⁺ strain. The sopB mutants also induced higher numbers of CD4⁺ CD44^hi CD62L^hi and CD8⁺ CD44^hi CD62L^hi central memory T cells. Eight weeks after primary oral immunization, mice were challenged with 100 50% lethal doses of virulent S. pneumoniae WU2. Immunization with either of the sopB mutant strains led to increased levels of protection compared to that with the isogenic sopB⁺ parent. Together, these results demonstrate that the deletion of sopB leads to an overall enhancement of the immunogenicity and efficacy of recombinant attenuated Salmonella vaccine strains.     

DNA vaccines expressing antigens with a stress protein-capturing domain display enhanced immunogenicity

Summary: An expression system for DNA vaccines is described, in which a fusion protein with an N‐terminal, viral J‐domain that captures heat‐shock proteins (Hsps) is translated in‐frame with C‐terminal antigen‐encoding sequences (of various lengths and origins). The system supports enhanced expression of chimeric antigens (of >800 residues in length) with an extended half life (>8 h). When used as a DNA vaccine, it delivers antigen together with the intrinsic adjuvant activity provided by bound Hsps. We describe the design of vectors for DNA vaccination that support the expression of different immunogenic domains of different origins as large, Hsp‐capturing chimeric fusion antigens. The immunogenicity of the antigens produced by this expression system (when it is built into DNA vaccines) has been characterized in detail, with particular emphasis on priming CD8⁺ T‐cell responses. We also discuss areas of vaccine research to which the new technology may provide useful contributions.