Genetic immunization of mice with DNA encoding the 23 kDa transmembrane surface protein of Schistosoma japonicum (Sj23) induces antigen-specific immunoglobulin G antibodies

The 23 kDa transmembrane surface protein of schistosomes is of recognized interest in studies of immune responsiveness in schistosomiasis. To examine the immunogenicity of the 23 kDa antigen of Schistosoma japonicum, Sj23, when delivered by genetic immunization, mice were immunized using a DNA construct containing the Sj23 cDNA under the control of a CMV promotor. Serological analysis of peripheral blood from immunized mice demonstrated that this construct was able to induce the production of antigen-specific IgG antibodies that recognized a schistosome antigen of 23 kDa in Western blots. Despite inducing antigen-specific antibodies, the Sj23 DNA vaccine was unable to confer protection in immunized mice subjected to challenge with S.japonicum cercariae. Appropriate engineering of the unique structure of the Sj23 kDa transmembrane protein of schistosomes may provide a novel vehicle for expressing foreign epitopes from other infectious agents or, possibly, cancer antigens, anchored to the surface of transfected cells.     

用噬菌体随机肽库鉴定日本血吸虫22.6kDa抗原分子的表位

目的　筛选和鉴定日本血吸虫 (中国大陆株 ) 2 2 .6kDa抗原 (Sj2 2 .6)的表位。　 方法　用纯化的抗Sj2 2 .6的多克隆抗体IgG对噬菌体十二肽库进行 5轮免疫学筛选 ,挑取克隆 ;采用Westernblotting免疫识别 ,将获得的阳性克隆免疫小鼠 ,并采用dot ELISA筛选能刺激小鼠产生较高滴度抗Sj2 2 .6抗体的阳性克隆 ;测定核苷酸序列 ,分析其表位与Sj2 2 .6抗原的同源性。　结果　经 5轮免疫学筛选后挑取的 1 4个克隆 ,用Westernblotting方法均能被抗Sj2 2 .6抗体识别。经动物免疫初步实验筛选 ,共获得 6个免疫原性较强的阳性克隆 ,测序结果获得 4种不同表位 ,其中 1种表位与Sj2 2 .6抗原具有较高的同源性 ,其余 3种表位与其无一级结构的同源性。　结论　获得的 4种日本血吸虫中国大陆株抗原表位 ,1种可能为结构表位 ,3种为模拟表位     

日本血吸虫(中国大陆株)22.6kDa抗原编码基因的核酸疫苗研究

为探索日本血吸虫（中国大陆株）２２．６ｋＤａ 抗原（Ｓｊ２２．６）编码基因用作核酸疫苗的可行性,将ｐＣＭＶ／Ｓｊ２２．６基因重组质粒经肌肉注射免疫了一批ＢＡＬＢ／ｃ小鼠并进行攻击感染试验,结果表明此重组质粒能在小鼠体内持续存在、稳定表达并诱导小鼠产生特异性的抗Ｓｊ２２．６ 抗体,但未能诱导有效的保护力     

Intradermal immunization of rats with plasmid DNA encoding Schistosoma mansoni 28 kDa glutathione S-transferase

Direct administration of plasmid DNA encoding an antigen represents an attractive approach to vaccination against infectious diseases, particularly in developing countries where easy-to-handle and cost-effective vaccines are needed. We have investigated the potential of DNA immunization to induce a specific antibody response against Schistosoma mansoni , using plasmid-DNA encoding the protective antigen, S. mansoni 28 kDa glutathione S-transferase (Sm28GST). Since S. mansoni parasite penetrates into its host through the skin, this tissue was chosen for plasmid DNA delivery. Following plasmid DNA administration into the skin of rats, the parasite antigen was detected in skin cells by immunohistochemistry. Three administrations of 200 μg plasmid at 14 day intervals led to the induction of a long-lasting specific IgG antibody response in the sera of immunized rats, with a predominance of IgG2a and IgG2b subclasses. Sera of immunized animals were able to mediate antibody-dependent cellular cytotoxicity in vitro , leading to the specific killing of parasite larvae. A parasite challenge performed on plasmid DNA-immunized animals induced a strong and rapid boosting effect on the specific IgG antibody response. These results demonstrate the potential of genetic immunization via the skin with plasmid DNA encoding Sm28GST for inducing immune responses with protective patterns against an S. mansoni infection .     

日本血吸虫中国大陆株基因重组抗原Sj 22.6kDa的核苷酸序列分析

目的：对ｐＧＳｊ２４克隆化基因进行核苷酸序列分析，了解其编码蛋白的属性。方法：常规制备ｐＧＳｊ２４克隆化基因并重组入测序载体Ｍ１３ｍｐ１９，以ＤＹＥＰＲＩＭＥＲ荧光测序试剂盒进行核苷酸序列测定。分别以ＤＮＡＳＩＳ和ＧＯＬＤＫＥＹ软件对序列资料进行分析。结果：ｐＧＳｊ２４克隆化基因长８４０ｂｐ，含一开放阅读框，可编码一分子量为２２．６ｋＤａ的蛋白质。开读框上游和下游均有终止密码子。该基因与已发表的日本血吸虫２２．６ｋＤａ蛋白的编码基因同源性达９５％，编码区同源性达９９．７％。在该基因内有一段典型的ＥＦ－Ｈａｎｄ钙结合区序列，并有内质网导肽、微体导向信号等功能位点。预测该蛋白质内可能的抗原决定簇位置为第２９－３２、６３－６８和８７－１０１等氨基酸片段。结论：ｐＧＳｊ２４克隆化基因为日本血吸虫２２．６ｋＤａ抗原编码基因。     

Production of IgE antibodies against the 22 kDa tegumental membrane-associated antigen of schistosomes is directed by the antigen itself

It has previously been reported that the predominant target of immunoglobulin E (IgE) recognition in sera from humans infected with Schistosoma japonicum in The Philippines or with S. mansoni in Kenya, is a 22 kDa tegumental membrane-associated schistosome antigen. In the present study, we demonstrate that the 22 kDa antigen can direct the production of antigen-specific IgE antibodies independently of schistosome infection and in the absence of any other parasite components or adjuvant. Three strains of mice were immunized using the purified, recombinant 22 kDa antigen of S. japonicum without the use of any adjuvant. Sera from all three strains of immunized mice, but not control animals, generated IgE antibodies specific for the native 22 kDa schistosome antigen in Western blots. Thus, the 22 kDa antigen itself must contain signals (presumably encoded by the primary amino acid sequence or by the secondary or tertiary structures of the molecule, or by a combination of these) which are sufficient to direct the isotype switch required for production of antigen-specific IgE .     

树突状细胞DNA多价疫苗抗日本血吸虫感染作用机制的研究

摘 要：目的 探讨树突状细胞（DC）DNA混合多价疫苗抗日本血吸虫感染保护性免疫作用机制。方法 BALB/c小鼠耳廓分别注射Sj26、Sj23和Sj14基因转染DC（A组）、Sj26基因转染DC（B组）、Sj23基因转染DC（C组）、Sj14基因转染DC（D组）、pcDNA3转染DC（E组）、未处理DC（F组）和RPMI-1640（G组）,共免疫3次,间隔2周,末次免疫后第2周,每鼠经皮肤感染40条日本血吸虫尾蚴。ELISA法检测血清特异性IgG抗体、干扰素-γ（IFN-γ）和白细胞介素-4（IL-4）水平,双夹心ELISA法检测脾淋巴细胞经ConA和可溶性虫卵抗原（SEA）刺激后培养上清中IFN-γ和IL-4水平,噻唑蓝（MTT）法检测脾淋巴细胞的增殖。结果 A组小鼠末次免疫后第2周血清特异性IgG抗体水平显著升高（与G组比较,P<0.001）。A组小鼠免疫后血清IFN-γ水平明显升高(P<0.01),而血清IL-4的水平,各组小鼠免疫前、后无明显变化。A组小鼠脾淋巴细胞经ConA和SEA刺激后诱生的IFN-γ水平显著增高,而IL-4水平显著降低(与G组比较,P<0.001)。A组小鼠脾淋巴细胞的刺激指数高于其他各组（与G组比较,P<0.001）。结论 体液免疫和细胞免疫共同参与了DC DNA混合多价疫苗诱导的保护性免疫作用,其中Th1型免疫应答在抗日本血吸虫感染的保护性免疫中起主要作用。 关键词：日本血吸虫;树突状细胞;DNA疫苗;保护性免疫     

Sj26和Sj23基因转染树突状细胞联合抗血吸虫感染的研究

目的探讨Sj26和Sj23基因转染树突状细胞(DC)联合抗日本血吸虫感染保护性免疫机制。方法BALB/c小鼠耳廓分别注射Sj26和Sj23基因转染DC(A组)、Sj26基因转染DC(B组)、Sj23基因转染DC(C组)、pcDNA3转染DC(D组)、未处理DC(E组)和RPMI-1640(F组),免疫3次,间隔2周,末次免疫后2周,每鼠经皮肤感染40条日本血吸虫尾蚴。ELISA法检测血清特异性IgG抗体、干扰素-γ(IFN-γ)和白细胞介素-4(IL-4)水平,双夹心ELISA法检测脾淋巴细胞经ConA和可溶性虫卵抗原(SEA)刺激后培养上清中IFN-γ和IL-4水平,噻唑蓝(MTT)法检测脾淋巴细胞增殖情况。结果末次免疫后第2周,A组小鼠血清IgG抗体水平显著升高(P<0.001)。血清IL-4的水平,各组免疫前、后无明显变化。血清IFN-γ水平,A组小鼠免疫后明显升高(P<0.01)。经ConA和SEA刺激后,A组小鼠脾淋巴细胞诱生的IFN-γ水平显著增高,而IL-4水平显著降低(与F组比较,P<0.001)。A组小鼠脾淋巴细胞经ConA和SEA刺激后的刺激指数均高于其他各组(与F组比较,P<0.001)。结论体液免疫和细胞免疫共同参与了Sj26和Sj23基因转染DC诱导的保护性免疫作用,其中Th1型免疫应答在抗日本血吸虫感染的保护性免疫中起主要作用。     

日本血吸虫(中国大陆株)22.6kDa抗原编码基因在C2C12细胞内的表达

目的为探索日本血吸虫（中国大陆株）22．6kDa抗原（Sj22．6）编码基因用作核酸疫苗的可行性。方法以PCR法对此编码基因改造后将其亚克隆入真核表达载体质粒PCMV－β并转化入大肠杆菌JM109进行大量扩增。将提纯的PCMV／Sj22．6基因重组质粒在体外转化C2C12真核细胞。结果免疫组化试验证明，该重组质粒能够在体外培养的C2C12细胞中表达Sj22.6抗原。结论表明该重组质粒有用作真核疫苗的可能性。     

Ro/SSA and La/SSB specific IgA autoantibodies in serum of patients with Sjögren's syndrome and systemic lupus erythematosus

OBJECTIVE: To investigate the occurrence of IgA autoantibodies to Ro 52 kDa, Ro 60 kDa and La antigen in serum of patients with primary Sj?gren's syndrome (pSS) and systemic lupus erythematosus (SLE). METHODS: Recombinant Ro 52 kDa, Ro 60 kDa and La antigens were used to analyse autoantibodies in serum from 25 patients with pSS, 30 patients with SLE and 20 controls using a semiquantitative immunoblotting approach. RESULTS: Among the patients with pSS, 21 (84%) had detectable IgA autoantibodies to Ro 52 kDa, 13 (52%) to Ro 60 kDa and 20 (80%) to La antigen. The corresponding results for the patients with SLE were 22 (73%), 14 (47%) and 20 (67%), respectively. No IgA autoantibodies against the three antigens were detected in 20 normal controls. A comparison of several clinical features with the titres of IgA antibodies to Ro 52 kDa, Ro 60 kDa and La, revealed a significant relation between IgA anti-Ro 52 and IgA anti-La to sicca (p< 0.05). Semiquantitative data suggest that IgG is the dominating antibody to the three antigens followed by IgM > IgA in both SLE and pSS patients. Specificity studies of IgA autoantibodies with different subfragments of Ro 52 kDa and Ro 60 kDa antigens showed that IgA antibodies did not differ from IgG and IgM in their recognition pattern. CONCLUSION: These results suggest that besides IgM and IgG, IgA autoantibodies are also detected at high frequency in patients with pSS and SLE. Further studies are necessary to evaluate the contribution of these IgA autoantibodies to inflammation as well as their diagnostic value.     

日本血吸虫22.6 kDa抗原编码基因序列中抑制性片段的确定

目的探讨日本血吸虫22.6kDa（Sj22.6）抗原编码基因序列中是否存在抑制性片段。方法用人工合成的来自Sj22.6抗原编码基因序列中的不同寡脱氧核苷酸和自小鼠脾脏分离的单个核细胞共同孵育,以对小鼠具有刺激作用的免疫刺激序列CpG1826作为刺激物,淋巴细胞增殖试验检测待测定寡脱氧核苷酸对CpG1826诱导的增殖是否具有抑制作用及其作用特征。结果存在于Sj22.6抗原编码基因序列中的寡脱氧核苷酸F311能够抑制刺激性CpG1826诱导的淋巴细胞增殖作用（P〈0.05）;当寡脱氧核苷酸F311与CpG1826的物质的量之比为1∶10和1∶3时无明显抑制作用（P〉0.05）,当物质的量之比为1∶1和3∶1时,对CpG诱导的淋巴细胞增殖的抑制率分别为11%和58%;寡脱氧核苷酸F311在先于CpG1826孵育2h加入表现出最强抑制作用。结论Sj22.6抗原编码基因序列中存在具有抑制作用的片段,抑制作用与其浓度和作用时间呈正相关。     

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.     

Self-replicative RNA vaccines elicit protection against influenza A virus, respiratory syncytial virus, and a tickborne encephalitis virus

In genetic vaccination, recipients are immunized with antigen-encoding nucleic acid, usually DNA. This study addressed the possibility of using the recombinant alpha virus RNA molecule, which replicates in the cytoplasm of transfected cells, as a novel approach for genetic vaccination. Mice were immunized with recombinant Semliki Forest virus RNA-encoding envelope proteins from one of 3 viruses: influenza A virus, a tickborne flavivirus (louping ill virus), or respiratory syncytial virus (RSV). Serologic analyses showed that antigen-specific antibody responses were elicited. IgG isotyping indicated that predominantly Th1 type immune responses were induced after immunization with RSV F protein-encoding RNA, which is relevant for protection against RSV infection. Challenge infection showed that RNA immunization had elicited significant levels of protection against the 3 model virus diseases.     

Echinococcus granulosus human infection stimulates low avidity anticarbohydrate IgG2 and high avidity antipeptide IgG4 antibodies

Total IgG and IgG subclasses recognizing carbohydrate and peptidic epitopes from native and periodate treated partially purified hydatid cyst fluid (ppHCFA) and protoscolex somatic antigens (PSA) were tested by ELISA in hydatid patients sera. Binding of the major cross-reactive antiphosphorylcholine antibodies was inhibited with free reagent. A predominant anticarbohydrate antibody response against ppHCFA and PSA is shown. Although the main contributing IgG subclass to the antipeptide response against both antigens was IgG4, IgG1 also significantly contributed to the anti-PSA peptidic epitopes response. Western blot showed that IgG1 antibodies strongly recognized in ppHCFA a periodate susceptible 38 kDa antigen. The IgG4 antibodies mainly recognized the periodate-resistant 12, 16 and 24 kDa antigens. In addition, IgG2 antibodies recognized three strongly periodate-susceptible broad bands (116, 55 and 24 kDa antigens). PSA-specific IgG1 and IgG4 antibodies showed similar patterns of antigen recognition as well as no significant reduction of reactivity after periodate treatment while the IgG2 antibody recognition was strongly affected by this treatment. Furthermore, IgG2 showed significantly lower avidities than IgG1 and IgG4 antibodies recognizing both antigens. In conclusion, hydatid patients showed an enhanced production of low avidity anticarbohydrate IgG2 as well as high avidity antipeptide IgG4 antibodies.     

Antibody isotype responses, infection and re-infection for Schistosoma japonicum in a marshland area of China.

Antibody isotype responses to adult worm antigen (AWA) of Schistosoma japonicum and two recombinant proteins (paramyosin (PMY) and a 22 kDa tegumental membrane-associated antigen (TEG)) were analyzed in 137 individuals from an area moderately endemic for schistosomiasis in the Dongting Lake region, Hunan Province, China. The prevalence and geometric mean (GM) intensity of infection before the implementation of curative chemotherapy were 28.5% and 234.4 epg, respectively, but 9 months after treatment the prevalence (6.6%) and intensity (38.3 epg) had decreased. There was no significant difference in either the prevalence or intensity of infection between males and females. Specific IgG (total), IgG4, IgG2, IgA and IgE responses to AWA, PMY and TEG were measured by ELISA. Males produced significantly (P < 0.05) more anti-AWA total IgG, IgE, IgA, IgG4 and IgG2 antibodies, and anti-TEG IgG2 antibody than their female counterparts. The OD450 levels of anti-AWA, PMY and TEG antibody isotypes did not present clear age-dependent trends except for peak levels of anti-AWA IgG4 antibodies evident among subjects 20-29 years of age. The total IgG and IgG4 antibody profiles against AWA correlated well with current S. japonicum infections while anti-AWA IgG2, IgA and IgE antibodies did not show such an association. Anti-AWA-specific IgE antibody levels were positively correlated (r = 0.55) with anti-AWA specific IgG4 antibody levels. In addition, the overall percentage of responders (using a cut-off value obtained from normal controls) to all isotypes to AWA were higher than those observed for both the recombinant antigens. Only 18.2%, 16.8% and 7.3% of the study population were IgE responders to AWA, PMY and TEG. A longer follow-up period is required before we can more fully understand the role of IgE, if any, in protective immunity against schistosomiasis japonica.     

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography–high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V_H CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V_H clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.     

Differential recognition of Loa loa antigens by sera of human subjects from a loiasis endemic zone

Somatic antigens of Loa loa adult worms with molecular weights of 15-180 kDa were identified by Western blot analysis using sera from 3 categories of parasitologically and clinically defined subjects from a loiasis endemic zone. Sera of occult, amicrofilaremic (OL), and 'resistant' individuals with no clinical signs of infection (R) reacted with an antigen of 160 kDa; sera of highly microfilaremic individuals (ML) did not. ML sera strongly reacted with an antigen of 18 kDa which was recognized only weakly or not at all by OL and R sera. At higher dilutions, OL sera only reacted with antigens at 23 and 160 kDa and ML sera reacted with antigens at 18 and 23 kDa, whereas R sera reacted with antigens at 23, 42, 54, 70, 100, and 160 kDa. These data suggested that R sera contained a higher concentration of antibodies which reacted with denatured, nitrocellulose-bound antigens. The IgG4 isotype predominated for all groups of sera, while IgG3 antibody responses were observed only with R sera. IgG1 antibodies were seen in all groups but reacted with fewer antigens than IgG4 antibodies, and no IgG2 antibody responses were detected. Sera against Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus, and Dirofilaria immitis cross-reacted with somatic antigens greater than 70 kDa, whereas none reacted with Loa loa antigens less than 23 kDa.     

日本血吸虫三类循环抗原和循环抗体对感染家兔疗效考核价值的研究

采用三株分别定位于日本血吸虫表皮膜、肠道上皮和虫卵的单克隆抗体A6,SJ31／32和SE9作为捕获抗体建立检测三类循环抗原的斑点酶联免疫吸附实验（DOT－ELISA）方法,并利用该方法和普通ELISA分别观察了日本血吸虫感染家兔血清中三类循环抗原和IgG抗体的动态变化情况。结果表明：感染日本血吸虫家免在经砒喹酮治疗之后的第8周,睦相关抗原,可溶性虫卵抗原和肠相关抗原的转阴率分别为80％,70％和20％,而减后第18周全部转阴;IgG抗体一直维持较高水平。因此,MAA和SEA具有改好的疗效考核价值,GAA的疗效考核效果较差,IgG抗体不具有疗效考核作用。     

Influence of adjuvants on murine immune responses against the C-terminal 19 kDa fragment of Plasmodium vivax merozoite surface protein-1 (MSP-1)

The immunogenicity of a yeast-expressed 19 kDa fragment of P vivax MSP-1 in the presence of different adjuvant formulations was evaluated. ICR mice were immunized with the 19 kDa antigen, using Freund's, alum, and block copolymer P1005 in water-in-oil (W/O) or oil-in-water (O/W) emulsions with or without detoxified lipopolysaccharide (RaLPS) as adjuvants. Five weeks following immunization with the antigen, mice were boosted with asexual blood-stage antigens. Three weeks after the last immunization with the 19 kDa antigen, mice from the Freund's group and most groups that received P1005 as adjuvant had higher total IgG titres than those that received alum as adjuvant or antigen alone. Antibody responses after the antigen immunization were predominantly of the IgG1 isotype, but mice in the Freund's and P1005 (W/O or O/W emulsion with or without RaLPS) groups also had high titres of IgG2a and IgG2b. Antibody titres against merozoites increased in all groups after the parasite antigen boost. IgG2a levels in the group that received antigen in P1005 plus RaLPS in the W/O emulsion were higher than those receiving Freund's, alum or the other copolymer adjuvants. The high IgG2a titres in this group were associated with reduced IL-10 production.     

Immunogenicity of HIV-1 Env and Gag in baboons using a DNA prime/protein boost regimen

OBJECTIVES: To evaluate the immunogenicity of sequence-modified HIV env and gag in baboons using DNA prime and protein boost strategy. METHODS: Synthetic sequence-modified HIV gene cassettes were constructed that expressed three different forms of Env proteins, gp140, gp140mut and gp140TM, plus or minus a mutation in the protease-cleavage site. These plasmids were used to immunize baboons (Papio cynocephalus). A group of baboons was also immunized with both env and gag DNA followed by p55Gag virus-like particles (VLP) boost. RESULTS: Modest antibody responses and low or no lymphoproliferative responses were observed following multiple DNA immunizations. In contrast, strong antibodies and substantial antigen-specific lymphoproliferative responses were seen following booster immunizations with oligomeric Env protein (o-gp140US4) in MF59. Neutralizing antibody responses were scored against T cell line adapted HIV-1 strains after the protein boosters, but neutralizing responses were low or absent against homologous and heterologous primary isolate strains. In the group receiving both gag and env vaccines, modest antigen-specific antibody and lymphoproliferative responses were scored after the DNA immunizations; these responses were enhanced several-fold upon boosting with the VLP preparations. The addition of Gag antigen did not interfere with Env-specific antibody responses, but there was a negative effect on the levels of Env-specific lymphoproliferation. CONCLUSIONS: These results highlight the importance of improving the potency of HIV DNA vaccines by enhanced DNA delivery and prime-boost vaccine technologies to generate more robust immune responses in larger animal models. In addition, care must be taken when immunizations with Env and Gag antigens are performed together.