A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.     

用重组腺病毒表达A组轮状病毒主要中和抗原VP7可获得良好的免疫学效果

为探索利用重组腺病毒表达轮状病毒的结构抗原以制备轮状病毒基因工程疫苗的可行性,构建了一株可表达A组轮状病毒主要中和抗原VP7的重组腺病毒AdEasyCVP7.AdEasyCVP7感染293细胞后,RT－PCR证明VP7基因有转,Western blotting试验可检测到VP7的表达。随后,用AdEasyCVP7通过灌胃和滴鼻两种不同途径免疫小鼠,并对免疫后小鼠的血清抗体和粘膜抗体进行了比较。初次免疫后,两组小鼠均有应答,但血清抗体滴度及阳转率不同。再次免疫后,滴鼻组小鼠显示出明显的加强效果。对肺灌洗液中的sIgA及肺、肠粘膜组织匀浆中的IgA进行检测发现滴鼻组的免疫学效果明显优于灌胃组。对血清中和抗体的检测表明,初次和再次免疫后,两组小鼠血清中均有中和抗体产生。该研究为轮状病毒基因工程疫苗的免疫方案、免疫途径及免疫保护作用等的进一步研究奠定了基础。     

Synthesis in Escherichia coli of the major glycoprotein of human rotavirus: analysis of the antigenic regions

Various regions of the gene encoding the major neutralization antigen, VP7, of human rotavirus have been expressed in Escherichia coli, as N-terminal fusions to beta-galactosidase under the control of the lac promoter. We have determined that the fusion products of two clones containing regions AB (aa 69-158) and ABC (aa 69-319) were antigenic, reacting with antibodies raised against whole virus. When guinea pigs were immunized with fusion protein purified by monoclonal antibody affinity columns, no neutralizing or virus-binding antibodies were detected, but antibodies binding to denatured VP7 were present.     

Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice

Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.     

Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus.

Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypically distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], less than 20). A reassortant (E19) that contained EDIM VP7 and WC3 VP4 was also very poorly neutralized by these antisera (GMT = 20). In contrast, antibody titers to a reassortant (R20) that contained WC3 VP7 and rhesus rotavirus VP4 were higher than those against WC3 (GMTs of 458 and 313, respectively). Thus, VP7 appeared to be the dominant immunogen for production of neutralizing antibody after intestinal infection of previously uninfected infants vaccinated with WC3 bovine rotavirus.     

嵌合型口蹄疫病毒样颗粒构建、表达及鉴定

为提高口蹄疫病毒(Foot-and-mouthdiseasevirus,FMDV)病毒样颗粒(Virus-likeparticles,VLPs)的特异性识别和递呈,为靶向疫苗研究奠定基础,利用反向PCR技术,将卵清蛋白(Ovalbumin,OVA)第257–264位氨基酸(Amino acids,aa)的短肽嵌入FMDV结构蛋白VP3第171–172位aa或第173–174位aa,通过大肠杆菌表达FMDV结构蛋白VP0、VP1和嵌合型VP3,体外组装得到嵌合OVA257-264肽的病毒样颗粒(VLPOVA)。用动态光散射、透射电镜检测VLPOVA大小和形态,免疫印迹、酶联免疫吸附试验和激光共聚焦显微镜检测短肽的嵌入情况。结果显示在VP3的第173–174位aa嵌入OVA,不影响蛋白表达和VLPs的组装且OVA位于VLPOVA的表面,VLPOVA粒径比VLPs稍大。     

Memory T-cell response to rotavirus detected with a gamma interferon enzyme-linked immunospot assay

Measurements of serum-neutralizing antibody and anti-rotavirus immunoglobulin A (IgA) are the current standard for assessing immune responses following rotavirus vaccination. However, there is ongoing debate as to whether antibody titers correlate with protection against rotavirus gastroenteritis. Children recovering from rotavirus gastroenteritis have increased gamma interferon release from cultured peripheral blood mononuclear cells (PBMCs), suggesting that cell-mediated immunity (CMI) may play a role in viral clearance and protection from subsequent gastroenteritis. We have developed a gamma interferon enzyme-linked immunospot (ELISPOT) assay for evaluation of CMI responses to rotavirus using frozen PBMCs obtained from healthy adults. Responses to three different rotavirus antigen types were analyzed-a peptide pool based on the human VP6 sequence; reassortant human:bovine vaccine strains; and cell culture-adapted (CCA) human G1, G2, G3, G4, and bovine (WC3) G6 strains. The reassortant strains consist of a bovine WC3 genome background expressing the human rotavirus surface proteins VP7 (G1, G2, G3, or G4) or VP4 (P1). Responses to titrations of the peptide pool as well as CCA and reassortant strains were assessed. Gamma interferon ELISPOT responses were similar for CCA and reassortant strains, whether live or UV inactivated, and when tested either individually or pooled. For most subjects, responses to the VP6 peptide pool positively correlated with responses to CCA and reassortant strains. Cell depletion studies indicate the memory responses detected with these frozen adult PBMCs were primarily due to the CD4+ T-cell population. This gamma interferon ELISPOT assay provides a new tool to apply in clinical studies for the characterization of natural or vaccine-induced CMI to rotavirus.     

Peroral immunization of microencapsulated human VP8 in combination with cholera toxin induces intestinal antibody responses

To develop an orally delivered subunit vaccine for rotavirus infection, a trypsin cleavage product of VP4, recombinant VP8*, was expressed in Escherichia coli. The recombinant VP8* (rVP8*), purified by affinity chromatography, was reactive against human rotavirus positive serum in Western-blot analysis. To further evaluate the immunogenicity of the oral-delivered rVP8*, it was encapsulated with alginate-microsphere and administered in combination with cholera toxin (CT) as a mucosal adjuvant perorally into mice. The ELISPOT assay showed that the number of rVP8*-specific IgG1 antibody secreting cells increased about 3-fold and about 2-fold in spleen and Peyer's patch, respectively as compared to non-immune mice. In addition, the number of rVP8*-specific IgA antibody secreting cells increased about 2-fold in Peyer's patch. Finally, rVP8*-specific IgA antibody response was significantly enhanced in the intestinal fluids from the mice immunized perorally with encapsulated rVP8* and CT. Taken together, these results indicate that rVP8* possessed proper immunogenicity and it would be potentially useful as a subunit vaccine against rotavirus-associated disease through peroral immunization.     

Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized （sVP6）. The VP6 and sVp6 genes were transformed into tobacco （Nicotiana tabacum L.） plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein （TSP）. Then, BALB/ c female mice that had been gavaged weekly with 10 mg TSP containing 34 p.g VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea.     

Antibody Responses to a Novel Plasmodium falciparum Merozoite Surface Protein Vaccine Correlate with Protection against Experimental Malaria Infection in Aotus Monkeys

The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals.     

展示生长抑素的猪细小病毒样颗粒构建及其免疫原性

为了获得既可预防猪细小病毒感染又能促进生长的嵌合病毒样颗粒疫苗,以PPV NJ-a株基因组DNA为模板扩增VP2基因片段,在VP2基因N端融合人工合成的4拷贝生长抑素基因,构建杆状病毒转移载体pFast-SS4-VP2。通过转化DH10Bac感受态细胞,pFast-SS4-VP2与穿梭载体Bacmid重组,获得重组Bacmid,命名为rBacmid-SS4-VP2。rBacmid-SS4-VP2转染Sf-9细胞,获得重组病毒rBac-SS4-VP2。SDS-PAGE与Western blotting鉴定可见约68 kDa的rSS4-VP2条带;rBac-SS4-VP2感染细胞IFA检测产生很强的特异性绿色荧光;感染细胞超薄切片电镜观察到大量特征性病毒样颗粒。将重组蛋白分别辅以铝胶、IMS和白油不同佐剂免疫小鼠,通过检测免疫小鼠VP2特异性ELISA抗体、PPV特异性中和抗体、生长抑素的抗体水平及生长激素水平来评价嵌合病毒样颗粒的免疫原性。结果表明,辅以铝胶与IMS佐剂重组蛋白组均产生了与PPV全毒组相似的ELISA抗体与中和抗体反应;重组蛋白免疫组均产生较好的针对生长抑素的抗体反应;免疫小鼠体内生长激素的水平明显升高;其中以铝胶佐剂组产生的各抗体水平最高,白油佐剂组各抗体水平最低。为以后生产安全、有效的颗粒化亚单位疫苗提供了一个新的设计思路,又为应用病毒样颗粒递呈外源肽,从而生产多联亚单位疫苗奠定了基础。     

Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector

Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.     

人轮状病毒ZTR-5株灭活疫苗的制备及小鼠血清免疫学评价

目的: 研究人轮状病毒ZTR-5株灭活疫苗的制备及在实验小鼠中的免疫原性评价。方法: 轮状病毒ZTR-5株在MA104细胞上经蚀斑筛选纯化后,获得单一克隆接种至Vero细胞上适应性培养,免疫荧光定量检测病毒的感染性滴度,对收获的病毒液进行离心、超滤、分子筛纯化,甲醛灭活,抗原定量检测Al(OH)₃吸附制备的实验性疫苗。使用不同剂量(8EU、32EU、128EU、256EU)经肌内注射免疫小鼠,共免疫三次,免疫间隔2周。采用间接ELISA法检测血清特异性抗体效价。 结果: 通过蚀斑纯化,筛选得到一株纯化的病毒株ZTR-5纯-1,在Vero细胞上适应性后感染性滴度达7.35logCCID₅₀/ml;大量培养收获的病毒原液滴度为7.57logCCID₅₀/ml,制备获得轮状病毒样品抗原含量为2 560EU/ml;经肌内注射,初次免疫后,所有剂量组动物均获得抗体阳转,阳转率为100%;第一次加强免疫后,各组血清特异性抗体水平均明显增高,免疫剂量为128EU和256EU的两组小鼠血清抗体效价均达1∶10 240;第二次加强免疫后,各剂量组(8EU、32EU、128EU、256EU)血清抗体效价依次达1∶5 120,1∶7 456,1∶14 481.54,1∶14 481.54。 结论:人轮状病毒ZTR-5株可在Vero细胞上稳定增殖,所制备的疫苗具良好免疫原性,用128EU/2次免疫即可获得良好的免疫效果。     

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.     

Mice develop effective but delayed protective immune responses when immunized as neonates either intranasally with nonliving VP6/LT(R192G) or orally with live rhesus rotavirus vaccine candidates

Rotavirus vaccines are delivered early in life, when the immune system is immature. To determine the effects of immaturity on responses to candidate vaccines, neonatal (7 days old) and adult mice were immunized with single doses of either Escherichia coli-expressed rotavirus VP6 protein and the adjuvant LT(R192G) or live rhesus rotavirus (RRV), and protection against fecal rotavirus shedding following challenge with the murine rotavirus strain EDIM was determined. Neonatal mice immunized intranasally with VP6/LT(R192G) were unprotected at 10 days postimmunization (dpi) and had no detectable rotavirus B-cell (antibody) or CD4(+) CD8(+) T-cell (rotavirus-inducible, Th1 [gamma interferon and interleukin-2 {IL-2}]-, Th2 [IL-5 and IL-4]-, or ThIL-17 [IL-17]-producing spleen cells) responses. However, by 28 and 42 dpi, these mice were significantly (P >or= 0.003) protected and contained memory rotavirus-specific T cells but produced no rotavirus antibody. In contrast, adult mice were nearly fully protected by 10 dpi and contained both rotavirus immunoglobulin G and memory T cells. Neonates immunized orally with RRV were also less protected (P=0.01) than adult mice by 10 dpi and produced correspondingly less rotavirus antibody. Both groups contained few rotavirus-specific memory T cells. Protection levels by 28 dpi for neonates or adults were equal, as were rotavirus antibody levels. This report introduces a neonatal mouse model for active protection studies with rotavirus vaccines. It indicates that, with time, neonatal mice develop full protection after intranasal immunization with VP6/LT(R192G) or oral immunization with a live heterologous rotavirus and supports reports that protection depends on CD4(+) T cells or antibody, respectively.     

Passive immunity to bovine rotavirus infection associated with transfer of serum antibody into the intestinal lumen.

The effect of circulating passive antibody on immunity to bovine rotavirus infections in neonatal calves was investigated. In the first experiment, rotavirus antibody titers in the small intestinal lumina of 5- and 10-day-old calves with a wide range of serum rotavirus antibody titers were determined. Neutralizing antibody was present in the small intestinal lumina in titers that correlated with the calves' serum titers (r = +0.84, P less than 0.01). Immunoglobulin G1 was the predominant isotype of intestinal luminal rotavirus antibody. Calves not fed colostrum during the absorptive period lacked rotavirus antibody in circulation and in the intestinal lumen at 7 days of age, even when they were fed large volumes of colostrum with a high rotavirus antibody titer at 48 h after birth. Therefore, rotavirus antibody is not retained in the intestinal lumen for 5 days following a colostrum meal, and the luminal antibody in the 5- and 10-day-old seropositive calves were probably derived from circulating antibody. In a second experiment, calves were passively immunized by subcutaneous injection of colostral whey with a high immunoglobulin G1 rotavirus antibody titer and challenged with virulent bovine rotavirus 48 h later. The passively immunized calves were protected from rotavirus infection and diarrhea compared with calves with comparable serum immunoglobulin concentrations but with lower serum rotavirus with lower serum rotavirus antibody titers. The results of these experiments indicate that circulating immunoglobulin G1 antibody appears in the gastrointestinal tract of neonatal calves and that circulating rotavirus antibody can prevent infection and diarrhea after rotavirus challenge.     

Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice

The VP8 subunit protein of human rotavirus (HRV) plays an important role in viral infectivity and neutralization. Recombinant peptide antigens displaying the amino acid sequence M(1)ASLIYRQLL(10), a linear neutralization epitope on the VP8 protein, were constructed and examined for their ability to generate anti-peptide antibodies and HRV-neutralizing antibodies in BALB/c mice. Peptide antigen constructs were expressed in E. coli as fusion proteins with thioredoxin and a universal tetanus toxin T-cell epitope (P2), in order to enhance the anti-peptide immune response. The peptide antigen containing three tandem copies of the VP8 epitope induced significantly higher levels of anti-peptide antibody than only a single copy of the epitope, or the peptide co-administered with the carrier protein and T-cell epitope. Furthermore, the peptide antigen containing three copies of the peptide produced significantly higher virus-neutralization titres, higher than VP8, indicating that a peptide antigen displaying repeating copies of the amino acid region 1-10 of VP8 is a more potent inducer of HRV-neutralizing antibodies than VP8 alone, and may be useful for the production of specific neutralizing antibodies for passive immunotherapy of HRV infection.     

Codelivery of the chemokine CCL3 by an adenovirus-based vaccine improves protection from retrovirus infection

Processing and presentation of vaccine antigens by professional antigen-presenting cells (APCs) is of great importance for the efficient induction of protective immunity. We analyzed whether the efficacy of an adenovirus-based retroviral vaccine can be enhanced by coadministration of adenovirus-encoded chemokines that attract and stimulate APCs. In the Friend retrovirus (FV) mouse model we coexpressed CCL3, CCL20, CCL21, or CXCL14 from adenoviral vectors, together with FV Gag and Env antigens, and then analyzed immune responses and protection from pathogenic FV infection. Although most tested chemokines did not improve protection against FV challenge, mice that received adenoviral vectors encoding CCL3 together with FV antigens showed significantly better control over viral loads and FV-induced disease than mice immunized with the viral antigens only. Improved protection correlated with enhanced virus-specific CD4+ T cell responses and higher neutralizing antibody titers. To apply these results to an HIV vaccine, mice were immunized with adenoviral vectors encoding the HIV antigens Env and Gag-Pol and coadministered vectors encoding CCL3. Again, this combination vaccine induced higher virus-specific antibody titers and CD4+ T cell responses than did the HIV antigens alone. These results indicate that coexpression of the chemokine CCL3 by adenovirus-based vectors may be a promising tool to improve antiretroviral vaccination strategies.     

Toward a poliovirus-based simian immunodeficiency virus vaccine: correlation between genetic stability and immunogenicity.

Recombinant polioviruses expressing foreign antigens may provide a convenient vaccine vector to engender mucosal immunity. Replication-competent chimeric viruses can be constructed by fusing foreign antigenic sequences to several positions within the poliovirus polyprotein. Artificial cleavage sites ensure appropriate proteolytic processing of the recombinant polyprotein, yielding mature and functional viral proteins. To study the effect of the position of insertion, two different recombinant polioviruses were examined. A small amino-terminus insertion delayed virus maturation and produced a thermosensitive particle. In contrast, insertion at the junction between the P1 and P2 regions yielded a chimeric poliovirus that replicated like the wild type. Eight different chimeras were constructed by inserting simian immunodeficiency virus (SIV) sequences at the P1/P2 junction. All recombinant viruses replicated with near-wild-type efficiency in tissue culture cells and expressed high levels of the SIV antigens. One of the inserted fragments corresponding to gp41 envelope protein was N-glycosylated but was not secreted. Inserted sequences were only partially retained after few rounds of replication in HeLa cells. This problem could be remedied to some extent by altering the sequences flanking the insertion point. Reducing the homology of the direct repeats by 37% decrease the propensity of the recombinant viruses to delete the insert. To determine the immunogenic potential of the recombinants, mice susceptible to poliovirus infection were inoculated intraperitoneally. The antibody titers elicited against Gag p17 depended on the viral doses and the number of inoculations. In addition, recombinants which display higher genetic stability were more effective in inducing an immune response against the SIV antigens, and inoculation with a mix of recombinants carrying different SIV antigens (a cocktail of recombinants) elicited humoral responses against each of the individual SIV sequences.     

Immunodominance of the VP4 neutralization protein of rotavirus in protective natural infections of young children.

Natural infection by very similar strains of rotavirus during the 1988-1989 rotavirus season in Cincinnati, Ohio, provided complete protection of young children against subsequent rotavirus illnesses for a period of at least 2 years. Using this limited strain variability, we characterized the association between the titers of antibody to either the VP4 or the VP7 neutralization protein and protection against subsequent rotavirus disease. This was done by using reassortants that contained only one of the two rotavirus neutralization proteins of 89-12, a culture-adapted isolate representative of the protective rotavirus strains. The other neutralization protein in these reassortants was derived from a heterologous rotavirus (WC3 or EDIM) to which the infected subjects made little or no neutralizing antibody (titers, < or = 20). The geometric mean titer (GMT) of antibody to 89-12 in convalescent-phase sera from the 21 subjects analyzed was 2,323. The GMT of antibody to a reassortant (strain WC-4) that contained the VP7 protein of 89-12 and VP4 of WC3 was 387. In contrast, the GMT of antibody to a reassortant (strain EDIM-7) that contained the VP4 protein of 89-12 and the VP7 protein of EDIM was 1,078. Thus, the major neutralization response was directed against VP4 rather than VP7, a finding that has important implications for development of appropriate rotavirus vaccines.