Immunogenicity of a recombinant protein containing the Plasmodium vivax vaccine candidate MSP1(19) and two human CD4+ T-cell epitopes administered to non-human primates (Callithrix jacchus jacchus)

One of the most promising vaccine candidates against the erythrocytic forms of malaria is the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)). As part of our studies aimed at the development of a Plasmodium vivax malaria vaccine, we characterized the immunogenic properties of a new bacterial recombinant protein containing the P. vivax MSP1(19) and two helper T-cell epitopes, the synthetic universal pan allelic DR epitope (PADRE) and a new internal MSP1 P. vivax epitope (DYDVVYLKPLAGMYK). We found that the recognition of His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE was as good as the recognition of His6MSP1(19) indicating that the presence of the T-cell epitopes PADRE and DYDVVYLKPLAGMYK did not modify the MSP1(19) epitopes recognized by human IgG. The recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE proved to be highly immunogenic in marmosets (Callithrix jacchus jacchus) when administered in incomplete Freund's adjuvant. However, when administered in other adjuvant formulations such as Quil A, CpG ODN 2006 or MPL/TDM, antibody titers to MSP1(19) were significantly lower. Among these three adjuvants, Quil A proved to be the most efficient one generating antibody titers significantly higher than the others. These results indicated that under the circumstances evaluated, adjuvants were key for the immunogenicity of the recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE.     

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

BACKGROUND: Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. METHODS: Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. RESULTS: Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). CONCLUSIONS: Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.     

Rectal immunization with rotavirus virus-like particles induces systemic and mucosal humoral immune responses and protects mice against rotavirus infection

To evaluate whether the rectal route of immunization may be used to provide appropriate protection against enteric pathogens such as rotaviruses (RV), we studied the antibody response and the protection induced by rectal immunization of mice with RV virus-like particles (VLP). For this purpose, 6-week-old BALBc mice were rectally immunized twice with RV 8-2/6/7-VLP derived from the bovine RV RF81 strain either alone or combined with various adjuvants including four toxins [cholera toxin (CT) and three attenuated Escherichia coli-derived heat-labile toxins (LTs), LT(R192G), LT(R72), and LT(K63)] and two Toll-like receptor-targeting adjuvants (CpG and resiquimod). Six weeks after the second immunization, mice were challenged with murine RV strain ECw. RV VLP administered alone were not immunogenic and did not protect mice against RV challenge. By contrast, RV VLP combined with any of the toxin adjuvants were immunogenic (mice developed significant titers of anti-RV immunoglobulin A [IgA] in both serum and feces and of anti-RV IgG in serum) and either efficiently induced complete protection of the mice (no detectable fecal virus shedding) or, for LT(K63), reduced the amount of fecal virus shedding after RV challenge. When combined with RV VLP, CpG and resiquimod failed to achieve protection, although CpG efficiently induced an antibody response to RV. These results support the consideration of the rectal route for the development of new immunization strategies against RV infection. Rectal delivery of a VLP-based vaccine might allow the use of adjuvants less toxic than, but as efficient as, CT.     

Antigenicity studies in humans and immunogenicity studies in mice: an MSP1P subdomain as a candidate for malaria vaccine development

The newly identified GPI-anchored Plasmodium vivax merozoite surface protein 1 paralog (MSP1P) has a highly antigenic C-terminus that binds erythrocytes. To characterize the antigenicity and immunogenicity of two regions (PvMSP1P-19 and -33) of the highly conserved C-terminus of MSP1P relative to PvMSP1-19, 30 P. vivax malaria-infected patients and two groups of mice (immunized with PvMSP1P-19 or -33) were tested for IgG subclass antibodies against PvMSP1P-19 and -33 antigens. In the patients infected with P. vivax, IgG1 and IgG3 levels were significantly higher than those levels in healthy individuals, and were the predominant response to the two C-terminal fragments of PvMSP1P (p < 0.05). In mice immunized with PvMSP1P-19, IgG1 levels were the highest while IgG2b levels were similar to IgG1 levels. The levels of Th1 cytokines in mice immunized with PvMSP1P-19 or -33 were significantly higher than those in mice immunized with PvMSP1-19 (p < 0.05). Our results indicate that: (i) IgG1 and IgG3 (IgG2b in mice) are predominant IgG subclasses in both patients infected with P. vivax and mice immunized with PvMSP1P-19 or -33; (ii) the C-terminus of MSP1P induces a Th1-cytokine response. This immune profiling study provides evidence that MSP1P may be a potential candidate for vivax vaccine.     

Rotavirus 2/6 Viruslike Particles Administered Intranasally with Cholera Toxin, Escherichia coli Heat-Labile Toxin (LT), and LT-R192G Induce Protection from Rotavirus Challenge

We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 μg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced equivalent geometric mean titers of rotavirus-specific serum antibody and intestinal immunoglobulin G (IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and LT-R192G were totally protected (100%) from rotavirus challenge, while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91% protection from challenge. The availability of a safe, effective mucosal adjuvant such as LT-R192G will increase the practicality of administering recombinant vaccines mucosally.     

Leishmania donovani recombinant iron superoxide dismutase B1 protein in the presence of TLR-based adjuvants induces partial protection of BALB/c mice against Leishmania major infection

In this study, we tested the protective efficacy of recombinant Leishmania donovani iron superoxide dismutase B1 (SODB1) against Leishmania major infection in BALB/c mice. Mice were challenged with L. major 3weeks after the second boost immunization with rSODB1 alone or in the presence of adjuvants. Injection of BALB/c mice with rSODB1 alone elicited both humoral and cellular immune responses. Administration of rSODB1 with CpG ODN or GLA-SE (a synthetic toll-like receptor 4 agonist) adjuvant resulted in the induction of anti-SODB1 IgG1, and more importantly of significantly high levels of IgG2a isotype. Immunization of mice with rSODB1 alone or with adjuvant induced the production of IFN-γ by splenocytes in response to stimulation with L. major soluble leishmanial antigens (SLA). Moreover, immunization protocols involving rSODB1 resulted in a significant decrease in IL-10 as compared to controls. The presence of CpG ODN or GLA-SE adjuvant in the immunization protocols resulted in a relative increase in IFN-γ in response to stimulation with rSODB1 in comparison to immunization with rSODB1 alone. Mice immunized with rSODB1 plus CpG ODN or GLA-SE, were able to partially control their Leishmania infections, as indicated by the reduction in footpad swelling and parasite numbers, compared to controls. These results suggest that immunization with recombinant SODB1 protein together with CpG ODN or GLA-SE can be potential vaccine candidate against leishmaniasis.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.     

CpG对乙型肝炎基因重组(CHO细胞)疫苗免疫效果的影响

为了研究CpG-寡脱氧核苷酸(CpG-OPN)作为佐剂对乙型肝炎基因重组(CHO细胞)疫苗(简称乙肝疫苗)免疫效果的影响,以乙肝疫苗加Al(OH)3、疫苗加CpG和疫苗加Al(OH)3与CpG3三种配伍方式,通过腹腔、皮下或肌内3种不同途径免疫Balb/c小鼠,观察不同免疫途径和不同配伍的免疫效果.同时又将疫苗与CpG混合后在4℃存放6个月再免疫小鼠,观察CpG的稳定性.结果表明:①3种免疫途径中以肌内注射效果最好,这在使用CpG的实验组尤为明显,在该组肌内免疫的ED50比腹腔的低了10倍,而诱发的抗体滴度提高了3倍;②疫苗与CpG、Al(OH)3联合使用的免疫效果最好,在肌内免疫时联合使用的免疫效果比疫苗+Al(OH)3提高4倍,比疫苗+CpG提高7倍;③疫苗+Al(OH)3免疫时,表现为IgG1抗体亚型占优势,而再加入CpG后则IgG1和IgG2a均升高,以IgG2a最显著;④疫苗与CpG混合后4℃保存半年,不影响其活性.     

Intranasal immunization of mice with CpG DNA induces strong systemic and mucosal responses that are influenced by other mucosal adjuvants and antigen distribution

BACKGROUND: Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory cytosine-guanine phosphate-linked dinucleotide (CpG) motifs are potent systemic and mucosal adjuvants in mice that have synergistic action with numerous other adjuvants, including alum and cholera toxin (CT). Herein, we evaluate CpG ODN with intranasal (IN) delivery of purified hepatitis B surface antigen (HBsAg), relative to and in combination with CT, Escherichia coli heat labile enterotoxin (LT), the B subunit of CT (CTB), and a nontoxic derivative of LT (LTK63). MATERIALS AND METHODS: BALB/c mice were immunized by IN administration of HBsAg, alone or combined with CT, LT, CTB, or LTK63, and/or CpG ODN, or non-CpG control ODN. In addition, the effect of low-or high-volume administration was assessed, in order to target upper respiratory or entire respiratory tract, respectively. HBsAg-specific systemic (immunoglobulins: IgG, IgG1, IgG2a in plasma) and mucosal (IgA in fecal, lung, vaginal, saliva, and gut samples) humoral responses, as well as cell-mediated immune responses including T-cell proliferation and cytokines (interleukins: IL-4, IL-5; interferon: IFN-gamma) were evaluated. RESULTS: CpG ODN, CT, and LT augmented anti-HBs titers equally, and more so than did CTB or LTK63. CpG ODN acted synergistically with CT and LT, but not CTB or LTK63 to enhance anti-HBs titers. Nevertheless, CpG ODN induced a more Th1-like response for all combinations, compared with the same formulation without CpG. Strength of induced systemic and mucosal immune responses was better with IN delivery of a large volume. A small volume required multiple administrations and higher doses of antigen and adjuvant for equal results. This suggests that delivery of antigen to the lung and/or diges-tive system is superior to delivery to the nasal cavity. CONCLUSIONS: Our results suggest that the synergy between CpG ODN and native toxins (CT, LT) may depend on their enzymatic activity and that the lack of synergy with nontoxic derivatives (LTB, LTK63) arises, since they do not have enzymatic activity. Because both CT and LT are too toxic for use in humans, it is possible that CpG ODN may be combined with bacterial toxin mutants that retain some enzymatic activity to optimize immune augmentation.     

Influence of adjuvants in inducing immune responses to different epitopes included in a multiepitope,multivalent, multistage Plasmodium falciparum candidate vaccine (FALVAC-1) in outbred mice

FALVAC-1, a vaccine against Plasmodium falciparum was developed by joining 21 epitopes from P. falciparum vaccine antigens and an universal T helper epitope from tetanus toxoid. Since adjuvants influence different aspects of immune responses, in this study we investigated the effect of four adjuvants aluminum hydroxide (alum), nonionic copolymer adjuvant P1005 (water-in-oil emulsion), CpG oligodeoxynucleotides (ODN), and QS-21 in eliciting immune responses in outbred mice. QS-21 and copolymer adjuvants were the best formulations in inducing higher and long-lasting antibody titers to the whole vaccine compared to alum and CpG. QS-21 was the only adjuvant to elicit predominantly IgG2a response and antibodies reactive with all epitopes incorporated in the vaccine construct. Vaccine elicited antibodies recognized sporozoites and asexual blood-stage parasites. FALVAC-1 immunized mice induced lymphoproliferative and IFN-gamma response to the vaccine. QS-21 and CpG adjuvants were able to elicit T proliferative responses to 20 of the 22 epitopes in the vaccine. In conclusion, this study demonstrated that with suitable adjuvant such as QS-21, it is possible to elicit immune responses to most of the epitopes included in the FALVAC-1 vaccine.     

Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA

Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein antigens administered by parenteral or mucosal routes to BALB/c mice. To date, there have been no studies using combined parenteral/mucosal approaches with CpG DNA as adjuvant. In this study we evaluated different parenteral prime-mucosal boost and mucosal prime-parenteral boost strategies using hepatitis B surface antigen (HBsAg) alone or with different adjuvants: aluminum hydroxide (alum), cholera toxin (CT), CpG ODN. In addition, since CpG ODN has previously been shown to act synergistically with other adjuvants after parenteral or mucosal delivery, we also evaluated adjuvant combinations: alum+CpG ODN and CT+CpG ODN. The effects of adjuvant and administration strategy on systemic and mucosal humoral responses were measured, as well as cell-mediated immune responses (cytotoxic T lymphocyte activity). These results were compared to parenteral only or mucosal only strategies. Our findings demonstrate that parenteral immunization can prime for mucosal responses even when different lymph nodes were being targeted. HBsAg-specific immune responses (IgG in plasma, cytotoxic T lymphocytes) induced by parenteral prime could all be significantly enhanced by mucosal boosting and despite the fact that intramuscular immunization alone could not induce mucosal IgA, it could prime for a subsequent mucosal boost. In addition, the presence of adjuvant at time of boosting could influence the nature of subsequent immune responses (Th1 vs. Th2). Mice primed intranasally could have their systemic immune responses boosted with a parenteral administration and it was also possible to enhance mucosal responses induced by intranasal prime with an intramuscular boost.     

Identification of the domains of the fibronectin-binding protein I of Streptococcus pyogenes responsible for adjuvanticity

Intranasal administration of antigens coupled to full-length fibronectin-binding protein I (SfbI) of Streptococcus pyogenes results in the elicitation of improved humoral and cellular immune responses, at both systemic and mucosal levels. We want to evaluate if SfbI also exhibits adjuvant properties when co-administered with the antigen, as well as identify the minimal domain responsible for its adjuvanticity. To achieve this aim, mice were immunized by the intranasal route with the model antigen beta-galactosidase (beta-gal) co-administered with recombinant proteins spanning different portions of the SfbI protein. The obtained results demonstrated that the adjuvant properties of SfbI were maintained intact when admixed to the model antigen. Similar kinetics and absolute titers of beta-gal-specific IgG antibodies as well as a dominant IgG(1) isotype response pattern were observed using SfbI derivatives spanning either the aromatic and proline-rich (H10) or the fibronectin-binding (H12) domains, respectively. The use of all tested derivatives also stimulated the elicitation of efficient beta-gal-specific IgA responses in lung lavages (23-25% of the total IgA). The obtained results suggest that different sub-domains of the SfbI protein can be used as adjuvants for the development of mucosal vaccines.     

Enhancement of mucosal immunization with virus-like particles of simian immunodeficiency virus

Cholera toxin (CT) is the most potent known mucosal adjuvant, but its toxicity precludes its use in humans. Here, in an attempt to develop safe and effective mucosal adjuvants, we compared immune responses to simian immunodeficiency virus (SIV) virus-like particles (VLPs) after intranasal coimmunization with RANTES, CpG oligodeoxynucleotides (ODN), or CT. Antibody analysis demonstrated that RANTES and CpG ODN had capacities for mucosal adjuvanticity, i.e., for enhancing serum and vaginal antibodies specific to SIV Env, similar to those for CT. RANTES and CpG ODN skewed serum antibodies predominantly to the immunoglobulin G2a isotype. Most importantly, RANTES and CpG ODN were more effective than CT in increasing neutralizing titers of both serum and vaginal antibodies. After intranasal coadministration with VLPs, RANTES or CpG ODN also induced increased levels of gamma interferon (IFN-gamma)-producing lymphocyte and cytotoxic T-lymphocyte activities in both spleen and lymph nodes but did not increase the levels of interleukin-4-producing lymphocytes. The results suggest that RANTES and CpG ODN enhance immune responses in a T-helper-cell-type-1 (Th1)-oriented manner and that they can be used as effective mucosal adjuvants for enhancing both humoral and cellular immune responses in the context of VLPs, which are particulate antigens.     

Immunogenicity of C-terminus of Plasmodium falciparum merozoite surface protein 1 expressed as a non-glycosylated polypeptide in yeast

The C-terminal region of the merozoite surface protein 1 (MSP1_(19)) is one of the mostpromising vaccine candidates against the erythrocytic forms of malaria.In the present study,a gene encodingPlasmodium falciparum MSP1_(19) was expressed in yeast Pichia pastoris.A non-glycosylated form of therecombinant protein MSP1_(19) was purified from culture medium.This recombinant protein maintains itsantigenicity.Significant immune responses were seen in C57BL/6 mice after the second immunization.Moreover,the specific antibodies recognized the native antigens of P.falciparum,The prevailing isotypesof immunoglobulin (Ig)G associated with immunization were IgG1,IgG2a and IgG2b.The antibodiesisolated from mouse sera immunized with MSP1_(19) can inhibit parasite growth in vitro.Based on theseimmunological studies,we concluded that MSP1_(19) deserves further evaluation in pre-clinical immunizationsagainst P.falciparum.     

不同佐剂对人乳头瘤病毒16型L2E7E6蛋白免疫效果的影响

目的:研究CpG佐剂、弗氏佐剂、聚肌胞苷酸佐剂及左旋咪唑、西米替丁作为佐剂对人乳头瘤病毒16型L2E7E6融合蛋白在小鼠体内产生的免疫效果的影响。方法:以单独蛋白组、蛋白加各佐剂组分别肌肉注射免疫C57BL/6小鼠,检测不同佐剂诱发小鼠产生的体液免疫和细胞免疫应答水平,并观察其对小鼠肿瘤生长的抑制作用。结果:各免疫组均能检测到高滴度的抗L2、E7、E6蛋白IgG抗体(以IgG1为主),其中弗氏佐剂能显著提高E6蛋白的IgG和IgG1抗体水平和E7蛋白的IgG1抗体水平(P<0.05),CpG佐剂明显提高了E7蛋白的IgG2a抗体水平(P<0.01);而西米替丁佐剂则降低了E7抗原的IgG抗体水平(P<0.05);同时可以检测到CpG佐剂组能诱发小鼠产生针对E7、E6较强的细胞免疫反应,且能抑制70%的荷瘤小鼠肿瘤生长;此外弗氏佐剂与聚肌胞苷酸佐剂可产生较弱的针对E7肽的细胞免疫反应,能延缓荷瘤小鼠肿瘤形成时间,与单纯蛋白组相比差异显著(P<0.05)。结论:CpG佐剂、弗氏佐剂和聚肌胞苷酸佐剂都能提高人乳头瘤病毒16型L2E7E6融合蛋白的细胞免疫反应水平和抑制肿瘤生长能力,其中CpG佐剂效果较好,为促进该蛋白作为疫苗的研发提供了实验依据。     

Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.     

Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines

Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-gamma]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.     

Cytokines as Adjuvants for the Induction of Anti-Human Immunodeficiency Virus Peptide Immunoglobulin G (IgG) and IgA Antibodies in Serum and Mucosal Secretions after Nasal Immunization

Safe and potent new adjuvants are needed for vaccines that are administered to mucosal surfaces. This study was performed to determine if interleukin-1alpha (IL-1alpha) combined with other proinflammatory cytokines provided mucosal adjuvant activity for induction of systemic and mucosal anti-human immunodeficiency virus (HIV) peptide antibody when intranasally administered with an HIV peptide immunogen. Nasal immunization of BALB/c mice with 10 microg of an HIV env peptide immunogen with IL-1alpha, IL-12, and IL-18 on days 0, 7, 14, and 28 induced peak serum anti-HIV peptide immunoglobulin G1 (IgG1) and IgA titers of 1:131,072 and 1:7,131, respectively (P = 0.05 versus no adjuvant). The use of cholera toxin (CT) as a mucosal adjuvant induced serum IgG1 and IgA titers of 1:32,768 and 1:776, respectively. The adjuvant combination of IL-1alpha, IL-12, and IL-18 induced anti-HIV peptide IgA titers of 1:1,176, 1:7,131, and 1:4,705 in saliva, fecal extracts and vaginal lavage, respectively. Titers induced by the use of CT as an adjuvant were 1:223, 1:1,176, and 1:675, respectively. These results indicate that the proinflammatory cytokines IL-1alpha, IL-12, and IL-18 can replace CT as a mucosal adjuvant for antibody induction and are important candidates for use as mucosal adjuvants with HIV and other vaccines.     

The effect of adjuvants on the efficacy of a recombinant herpes simplex virus glycoprotein vaccine

A recombinant, truncated HSV type 1 glycoprotein D secreted by Chinese hamster ovary cells (rgD1) was used to compare the ability of several adjuvants to stimulate protective immunity in guinea pigs. Adjuvants tested included CFA, aluminum hydroxide (alum), a lipophilic derivative of muramyl tripeptide (MTP-PE), and a muramyl dipeptide (MDP) covalently conjugated to rgD1. Animals were immunized three times with rgD1 plus the various adjuvants and antibody titers were determined by ELISA. Four weeks after the last immunization, the animals were challenged intravaginally with HSV type 2 and were monitored daily for clinical signs of disease, including frequency and severity of herpetic lesions, incidence of urinary retention, and mortality during the 14-day post-challenge observation period. Animals immunized in the foot-pad with rgD1 formulated with CFA showed the highest antibody titers. Animals immunized in the footpad with rgD1 using MTP-PE in a 4% squalene formulation, alum, or rgD1 conjugated to MDP showed mean antibody titers that were 57, 16, and 13% of the CFA titers, respectively. Immunization with rgD1 plus MTP-PE, alum, or rgD1-MDP conjugate by the i.m. route elicited lower antibody titers than the footpad route of immunization. Results of the viral challenge indicated that clinical symptoms of the groups immunized with rgD1 with CFA or MTP-PE as adjuvant were similar in magnitude and were markedly reduced compared with unimmunized control groups. Animals immunized with rgD1 combined with alum or rgD1-MDP conjugate showed clinical symptoms significantly more severe than the CFA or MTP-PE groups. The protective immunity observed after i.m. immunization of animals with rgD1 and MTP-PE was only slightly lower than animals immunized with the same Ag-adjuvant combination in the footpad. The results indicate that MTP-PE is an effective adjuvant for the recombinant herpes gD vaccine.     

不同佐剂条件下Aβ多肽B细胞表位疫苗诱导产生抗体的免疫反应特性分析

目的:研究阿尔茨海默病β淀粉样肽(Aβ)B细胞表位疫苗2Aβ1-15-PADRE(Aβ-T)诱导产生抗体的免疫反应特性,并探讨不同佐剂对该疫苗免疫反应效果的影响。方法:合成了含2个Aβ42的 B细胞表位—Aβ1-15及1个辅助T细胞表位—PADRE的多肽2Aβ1-15-PADRE。采用Al(OH)₃佐剂,弗氏佐剂,Abisco佐剂,MF59佐剂分别与多肽疫苗联合免疫小鼠,并另设3个对照组:无佐剂多肽免疫组(Mock),PBS免疫组(PBS),未免疫组(Native)。结果:5组多肽免疫组小鼠均产生了针对Aβ的特异性抗体,无佐剂多肽免疫组的IgG抗体滴度最低,Al(OH)₃佐剂组,MF59佐剂组,Abisco佐剂组小鼠IgG抗体滴度较高,弗氏佐剂组IgG抗体滴度最高。斑点杂交实验结果显示5组小鼠免疫后血清与Aβ42单体反应较弱,与寡聚体反应最明显,与纤维状Aβ42几乎不反应。结论:4种佐剂均能提高多肽疫苗的免疫反应,产生高水平抗Aβ的特异性抗体。5组免疫小鼠产生的抗体均与Aβ寡聚体反应较强,与纤维状Aβ42反应较弱,表明该多肽疫苗具有良好的应用前景。