Response of primed human PBMC to synthetic peptides derived from hepatitis B virus envelope proteins: a search for promiscuous epitopes

This investigation was aimed at identifying effective T helper cell epitopes to the hepatitis B virus in humans. A panel of synthetic peptides that represent the hepatitis B virus whole envelope proteins was examined for their capability to stimulate peripheral blood mononuclear cells from human subjects infected with hepatitis B virus naturally. In addition, a large number of subjects were examined and their human leukocyte antigen (HLA) class II allele types were identified to determine whether the helper T cell epitope is specific for a particular HLA allele or 'promiscuous'. The peptides of the amino acid residues 52-67, 110-125, 190-205, and 228-243 appeared to be immunogenic, and particularly, the 52-67 residue was the most promiscuous epitope peptide. These results would contribute to the better understanding of the helper T cell responses to the hepatitis B virus and provide a useful way in designing epitope-based vaccines and future therapeutic strategies.     

"Negative vaccination" by specific CD4 T cell tolerisation enhances virus-specific protective antibody responses

Background

Cooperation of CD4⁺ T helper cells with specific B cells is crucial for protective vaccination against pathogens by inducing long-lived neutralizing antibody responses. During infection with persistence-prone viruses, prolonged virus replication correlates with low neutralizing antibody responses. We recently described that a viral mutant of lymphocytic choriomeningitis virus (LCMV), which lacks a T helper epitope, counterintuitively induced an enhanced protective antibody response. Likewise, partial depletion of the CD4⁺ T cell compartment by using anti-CD4 antibodies enhanced protective antibodies.

Principal Findings

Here we have developed a protocol to selectively reduce the CD4⁺ T cell response against viral CD4⁺ T cell epitopes. We demonstrate that in vivo treatment with LCMV-derived MHC-II peptides induced non-responsiveness of specific CD4⁺ T cells without affecting CD4⁺ T cell reactivity towards other antigens. This was associated with accelerated virus-specific neutralizing IgG-antibody responses. In contrast to a complete absence of CD4⁺ T cell help, tolerisation did not impair CD8⁺ T cell responses.

Conclusions

This result reveals a novel “negative vaccination” strategy where specific CD4⁺ T cell unresponsiveness may be used to enhance the delayed protective antibody responses in chronic virus infections.     

Mapping of B-neutralizing and T-helper cell epitopes on the bovine leukemia virus external glycoprotein gp51.

A battery of 19 synthetic peptides was used to characterize efficient neutralizing and helper T-cell epitopes on the bovine leukemia virus (BLV) external envelope glycoprotein gp51. Four of the antipeptide antisera raised in rabbits inhibited the formation of BLV-induced syncytia; these antisera are directed against peptides 64-73, 98-117, and 177-192. Only antisera directed against the 177-192 region also neutralized vesicular stomatitis virus-BLV pseudotypes. This study clearly demonstrates that neutralizing properties can be observed with antibodies raised to regions undescribed so far and included in both the amino-terminal and central parts of the antigen. In addition, some helper T-cell determinants were defined from gp51-immunized mice and from BLV-infected cattle. Although none of the peptides tested behaved as a universal helper T-cell epitope, peptide 98-117 stimulated T-cell proliferation from BALB/c mice and from three infected cows, while peptide 169-188 strongly stimulated T-cell proliferation from one infected cow. Further experiments performed with three peptides overlapping the 169-188 region (177-192, 179-192, 181-192) demonstrated the particular relevance of residue(s) P-177 and/or D-178 in the helper T-cell epitope. These data should assist in the design of an efficient subunit vaccine against BLV infection that contains peptides possessing both B-neutralizing and helper T-cell determinants.     

The anamnestic neutralizing antibody response is critical for protection of mice from challenge following vaccination with a plasmid encoding the Japanese encephalitis virus premembrane and envelope genes.

For Japanese encephalitis (JE), we previously reported that recombinant vaccine-induced protection from disease does not prevent challenge virus replication in mice. Moreover, DNA vaccines for JE can provide protection from high challenge doses in the absence of detectable prechallenge neutralizing antibodies. In the present study, we evaluated the role of postchallenge immune responses in determining the outcome of JE virus infection, using mice immunized with a plasmid, pcDNA3JEME, encoding the JE virus premembrane (prM) and envelope (E) coding regions. In the first experiment, 10 mice were vaccinated once (five animals) or twice (remainder) with 100 micrograms of pcDNA3JEME. All of these mice showed low (6 of 10) or undetectable (4 of 10) levels of neutralizing antibodies. Interestingly, eight of these animals showed a rapid rise in neutralizing antibody following challenge with 10,000 50% lethal doses of JE virus and survived for 21 days, whereas only one of the two remaining animals survived. No unimmunized animals exhibited a rise of neutralizing antibody or survived challenge. Levels of JE virus-specific immunoglobulin M class antibodies were elevated following challenge in half of the unimmunized mice and in the single pcDNA3JEME-immunized mouse that died. In the second experiment, JE virus-specific primary cytotoxic T-lymphocyte (CTL) activity was detected in BALB/c mice immunized once with 100 micrograms of pcDNA3JEME 4 days after challenge, indicating a strong postchallenge recall of CTLs. In the third experiment, evaluation of induction of CTLs and antibody activity by plasmids containing portions of the prM/E cassette demonstrated that induction of CTL responses alone were not sufficient to prevent death. Finally, we showed that antibody obtained from pcDNA3JEME-immunized mice 4 days following challenge could partially protect recipient mice from lethal challenge. Taken together, these results indicate that neutralizing antibody produced following challenge provides the critical protective component in pcDNA3JEME-vaccinated mice.     

Priming of influenza virus-specific cytotoxic T lymphocytes vivo by short synthetic peptides.

Influenza virus-specific CTL were primed in vivo by immunization with short synthetic peptides representing major CTL epitopes from the nucleoprotein of type A influenza virus. The resultant CTL after in vitro boosting of primed spleen cells recognized both virus-infected and peptide-pulsed target cells. The requirement of CD4+ T cell activation was investigated in several ways. First the addition of helper epitopes to the CTL epitope did not enhance CTL generation, suggesting that helper activity was either not limiting or not required. However, in vivo depletion of CD4+ T cells completely inhibited the generation of CTL by peptide immunization. The inclusion of anti-CD4 in the in vitro restimulation with peptide also prevented the generation of CTL, whereas in vitro reactivation of virus immune spleen cells with peptide was not inhibited by anti-CD4. Thus there appears to be heterogeneity in the requirement of CD4+ T cell proliferation in CTL generation. One possibility is that virus infected cells can stimulate higher affinity T cells that are less helper T cell dependent.     

Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang,an Eastern Province of China

BackgroundSporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains.Conclusion/SignificancesJE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.     

Preparation of a purified, inactivated Japanese encephalitis (JE) virus vaccine in Vero cells

An attenuated Japanese encephalitis (JE) virus SA14-14-2 (PDK) was adapted to Vero cells, a continuous cell line that has been licensed for human vaccine production, by serial passages. The resulting virus was purified by tangential flow ultrafiltration followed by sucrose density gradient ultracentrifugation, giving 2.3 mg purified virus per liter of culture supernatant. Treatment with 0.05% formalin for 4 days at 22 °C completely inactivated viral infectivity while preserving its antigenicity. The purified, inactivated JE virus was formulated with alum hydroxide and administered to mice by intraperitoneal route. In terms of its ability to induce anti-JE neutralizing antibody and to protect the immunized animal against neurovirulent virus challenge, the purified, inactivated JE virus formulated with alum was equivalent to the exiting commercial mouse brain-derived vaccine (JE-VAX, Aventis Pasteur Inc.).     

Risk of immunodeficiency virus infection may increase with vaccine-induced immune response

To explore the efficacy of novel complementary prime-boost immunization regimens in a nonhuman primate model for HIV infection, rhesus monkeys primed by different DNA vaccines were boosted with virus-like particles (VLP) and then challenged by repeated low-dose rectal exposure to simian immunodeficiency virus (SIV). Characteristic of the cellular immune response after the VLP booster immunization were high numbers of SIV-specific, gamma interferon-secreting cells after stimulation with inactivated SIV particles, but not SIV peptides, and the absence of detectable levels of CD8(+) T cell responses. Antibodies specific to SIV Gag and SIV Env could be induced in all animals, but, consistent with a poor neutralizing activity at the time of challenge, vaccinated monkeys were not protected from acquisition of infection and did not control viremia. Surprisingly, vaccinees with high numbers of SIV-specific, gamma interferon-secreting cells were infected fastest during the repeated low-dose exposures and the numbers of these immune cells in vaccinated macaques correlated with susceptibility to infection. Thus, in the absence of protective antibodies or cytotoxic T cell responses, vaccine-induced immune responses may increase the susceptibility to acquisition of immunodeficiency virus infection. The results are consistent with the hypothesis that virus-specific T helper cells mediate this detrimental effect and contribute to the inefficacy of past HIV vaccination attempts (e.g., STEP study).     

In vivo induction of cytotoxic T cell response by a free synthetic peptide requires CD4+ T cell help.

Most attempts to induce CTL responses by in vivo priming with free synthetic peptides have been unsuccessful so far. However, two separate studies have recently succeeded in inducing antiviral CTL responses by immunizing mice with unmodified free synthetic peptides derived from nucleoproteins from either lymphocytic choriomeningitis virus or Sendai virus. In the present study, we have analyzed the cellular mechanisms by which the lymphocytic choriomeningitis virus synthetic peptide induced CTL responses. We demonstrated that this peptide, which was previously shown to be recognized by CD8+ T cells, also contains a helper CD4+ T cell epitope. It stimulates in vivo both CD4+ T cell-mediated CTL response. The in vivo elimination of CD4+ T cells by treatment with a mAb was shown to strongly reduce the antipeptide CTL response. This study therefore demonstrates that to be able to induce CTL responses, a peptide has to stimulate both CD4+ and CD8+ T cell subset.     

Heterogeneity in the recognition of the simian immunodeficiency virus envelope glycoprotein by CD4+ T cell clones from immunized macaques.

CD4+ T cell recognition of the simian immunodeficiency virus (SIV) surface envelope (env) glycoprotein was examined by using a panel of 10 T cell lines and 4 T cell clones derived from 10 individual macaques immunized with inactivated SIV or recombinant SIV env proteins. The results demonstrated that CD4+ T cells from each animal recognized between 1 and 7 peptides in 4 distinct regions of the protein including both variable and conserved domains. MLR of PBMC from selected macaques together with RFLP analysis by using the HLA DR beta probes suggested that animals of distinct MHC class II haplotypes can recognize identical peptides. These T cell epitopes within conserved regions of the envelope protein, together with identified linear B cell epitopes recognized by neutralizing antibodies, may be relevant in vaccine design.     

Polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120 T helper cell sites and B cell neutralization epitopes

In previous studies, we have used antisera raised to envelope (env)-gene-encoded synthetic peptides to identify a region of (HIV) glycoprotein (gp) 120 env protein designated SP10 that contains a type-specific neutralizing determinant. To develop a polyvalent, synthetic peptide inoculum that can evoke both neutralizing antibodies and T cell proliferative responses to more than one HIV isolate, synthetic peptides containing type-specific neutralizing determinants of gp120 from HIV isolates HTLV-IIIB (IIIB), HTLV-IIIMN (MN) and HTLV-IIIRF (RF) were coupled to a 16 amino acid T cell epitope (T1) of HIV-IIIB gp120 and used to immunize goats. Goat antisera to each T1-SP10 peptide derived from the SP10 region of gp120 of IIIB, MN, and RF neutralized HIV isolates IIIB, MN and RF in a type-specific manner. Moreover, peripheral blood T cells from immunized goats also proliferated in a type-specific manner to peptides derived from gp120 of IIIB, MN, and RF. When combined in a trivalent inoculum, T1-SP10 peptides from HIV-1 isolates IIIB, MN, and RF evoked a high titered neutralizing antibody response to isolates IIIB, MN, and RF in goats and as well induced immune T cells to undergo blast transformation in the presence of peptides derived from gp120 of all three HIV isolates. The T1 portion of the T1-SP10 construct was shown to induce a B cell antibody response against determinants within the T1 peptide in addition to inducing T cell proliferative responses in immune goat T cells. Moreover, the SP10 portion of the T1-SP10 constructs not only induced B cell antibody production but also induced type-specific T cell proliferative responses localized to the C-terminal variable sequences of the SP10 peptides. Finally, the T1-SP10 peptide construct induced memory T cell proliferative responses to native gp120 env protein. Thus, combinations of homologous SP10 region synthetic peptides containing type-specific neutralizing determinants and T cell epitopes of HIV gp120 may be useful in man to elicit high titered neutralizing B cell responses and, as well, T cell responses to more than one HIV isolate.     

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. I. Helper cells have a more restricted specificity repertoire than proliferative cells

The proliferative and helper T cell repertoires were compared in the CBA/J mouse for the response to the large protein antigen, tetrameric beta-galactosidase (GZ = 1021 a.a/monomer). The systems assessed the ability of cyanogen bromide (CB) peptides of GZ to: 1) prime for a T cell proliferative response to GZ; or 2) generate T cell help, measured by the production of anti-FITC PFC in the in vitro response to GZ-FITC. Priming for in vitro proliferation was attempted with 11 CB peptides comprising 70% of the GZ molecule. Strong priming was found with five peptides and intermediate priming was found with four other peptides; two peptides were without effect (CB-20 = a.a 767-862, and CB-4 = a.a. 188-202). Despite this indication of generally dispersed recognition of GZ epitopes, only two CB peptides, CB-2 (a.a. 3-92) and CB-10 (a.a. 378-418) were able to induce a T helper cell response. The surprising dearth of helper T cell-inducing epitopes may be peculiar to the limited fluorescein (FITC) substitution on GZ-FITC (17-25 FITC residues per tetramer) or it may reflect the constraints involved in T cell recognition required for T-B collaboration. Also considered was the possibility that the helper T cell repertoire might be distinct from the proliferative repertoire, the latter reflecting DNA synthesis and recruitment by other functional T cell subpopulations.     

Antigenic analysis of flaviviruses with monoclonal antibodies against Negishi virus

Twelve monoclonal antibodies against Negishi virus were obtained and characterized by hemagglutination inhibition (HI) and neutralization (NT) tests using five flaviviruses isolated in the pan-Pacific region. The reaction pattern of the antibodies showed that Negishi virus was most closely related to Langat virus, followed by 3-Arch, JE and Apoi viruses in that order. Hemagglutinating (HA) antigen of the virus had distinct HI relating sites which were Negishi virus specific, tick borne encephalitis (TBE) virus complex specific and flavivirus cross-reactive. Monoclonal anti-Negishi antibodies cross-reactive to Japanese encephalitis (JE) virus in the HI test had neutralizing activity to JE virus but no activity to homologous Negishi virus.     

Role of receptor-binding activity of the viral hemagglutinin molecule in the presentation of influenza virus antigens to helper T cells.

The concentration of antigen required to stimulate influenza virus-specific helper T cells was observed to be dependent upon the antigenic form bearing the relevant determinant: intact, nonreplicative virus was needed only in picomolar amounts, while denatured proteins, protein fragments, or synthetic peptides were required in micromolar concentrations for a threshold level of stimulation. Antigenic efficiency of intact virus was found to result from the attachment of virus to sialic acid residues on the surface of the antigen-presenting cell since spikeless viral particles lacking the hemagglutinin molecule were much less efficient antigens for helper T cells and continuous presence of hemagglutination-inhibiting antihemagglutinin antibodies reduced efficiency of stimulation by intact virus approximately 100-fold for both hemagglutinin and internal virion proteins. Influenza virus associated rapidly with antigen-presenting cells; less than 10 min at 20 degrees C was sufficient to introduce virus for a maximal level of T-cell stimulation. This rapid attachment was blocked by antibodies to the hemagglutinin or by pretreatment of the antigen-presenting cells with neuraminidase to remove the cellular virus receptor. Following viral adsorption by antigen-presenting cells, a lag period of 30 min at 37 degrees C was required for the expression of helper T-cell determinants. One early event identified was the movement of the virus to a neuraminidase-insensitive compartment, which can occur at 10 degrees C, but which was not equivalent to expression of helper T-cell determinants. Preincubation of cells with virus at 10 degrees C for 4 h reduced the lag period of helper T-cell determinant expression to 15 min when these cells were shifted to 37 degrees C, suggesting that transition of the virus to a neuraminidase-resistant state is a required step in presentation of T-cell antigenic determinants.     

T-helper cell and associated antibody response to synthetic peptides of the E glycoprotein of Murray Valley encephalitis virus.

A battery of 16 synthetic peptides, selected primarily by computer analysis for predicted B- and T-cell epitopes, was prepared from the deduced amino acid sequence of the envelope (E) glycoprotein of Murray Valley encephalitis (MVE) virus. We examined all of the peptides for T-helper (Th)-cell recognition and antibody induction in three strains of mice: C57BL/6, BALB/c, and C3H. Lymphoproliferative and interleukin-2 assays were performed on splenic T cells from mice inoculated with peptides in Freund's incomplete adjuvant or with MVE virus. Several peptides found to contain predicted T-cell epitopes elicited a Th-cell response in at least one strain of mice, usually with a concomitant antibody response. Peptides 145 (amino acids 145 to 169) and 17 (amino acids 356 to 376) were strongly recognized by T cells from all three inbred strains of mice. Peptide 06 (amino acids 230 to 251) primed C57BL/6 mice for Th- and B-cell reactivity with native MVE virus, and T cells from virus-immune mice were stimulated by this peptide. Peptide 06 was recognized by several Th-cell clones prepared from mice immunized with MVE, West Nile, or Kunjin virus. These results indicate that it may be feasible to design synthetic flavivirus peptides that define T-cell epitopes capable of generating a helper cell response for B-cell epitopes involved in protective immunity.     

Extralymphatic virus sanctuaries as a consequence of potent T-cell activation

T helper cells can support the functions of CD8(+) T cells against persistently infecting viruses such as murine lymphocytic choriomeningitis virus (LCMV), cytomegalovirus, hepatitis C virus and HIV. These viruses often resist complete elimination and remain detectable at sanctuary sites, such as the kidneys and other extralymphatic organs. The mechanisms underlying this persistence are not well understood. Here we show that mice with potent virus-specific T-cell responses have reduced levels and delayed formation of neutralizing antibodies, and these mice fail to clear LCMV from extralymphatic epithelia. Transfer of virus-specific B cells but not virus-specific T cells augmented virus clearance from persistent sites. Virus elimination from the kidneys was associated with the formation of IgG deposits in the interstitial space, presumably from kidney-infiltrating B cells. CD8(+) T cells in the kidneys of mice that did not clear virus from this site were activated but showed evidence of exhaustion. Thus, we conclude that in this model of infection, site-specific virus persistence develops as a consequence of potent immune activation coupled with reductions in virus-specific neutralizing antibodies. Our results suggest that sanctuary-site formation depends both on organ anatomy and on the induction of different adaptive immune effector mechanisms. Boosting T-cell responses alone may not reduce virus persistence.     

Immune and viral correlates of "secondary viral control" after treatment interruption in chronically HIV-1 infected patients

Upon interruption of antiretroviral therapy, HIV-infected patients usually show viral load rebound to pre-treatment levels. Four patients, hereafter referred to as secondary controllers (SC), were identified who initiated therapy during chronic infection and, after stopping treatment, could control virus replication at undetectable levels for more than six months. In the present study we set out to unravel possible viral and immune parameters or mechanisms of this phenomenon by comparing secondary controllers with elite controllers and non-controllers, including patients under HAART. As candidate correlates of protection, virus growth kinetics, levels of intracellular viral markers, several aspects of HIV-specific CD4+ and CD8+ T cell function and HIV neutralizing antibodies were investigated. As expected all intracellular viral markers were lower in aviremic as compared to viremic subjects, but in addition both elite and secondary controllers had lower levels of viral unspliced RNA in PBMC as compared to patients on HAART. Ex vivo cultivation of the virus from CD4+ T cells of SC consistently failed in one patient and showed delayed kinetics in the three others. Formal in vitro replication studies of these three viruses showed low to absent growth in two cases and a virus with normal fitness in the third case. T cell responses toward HIV peptides, evaluated in IFN-γ ELISPOT, revealed no significant differences in breadth, magnitude or avidity between SC and all other patient groups. Neither was there a difference in polyfunctionality of CD4+ or CD8+ T cells, as evaluated with intracellular cytokine staining. However, secondary and elite controllers showed higher proliferative responses to Gag and Pol peptides. SC also showed the highest level of autologous neutralizing antibodies. These data suggest that higher T cell proliferative responses and lower replication kinetics might be instrumental in secondary viral control in the absence of treatment.     

Identification and characterization of T helper epitopes in the nucleoprotein of influenza A virus

By using a series of overlapping synthetic peptides that cover more than 95% of the amino acid sequence of nucleoprotein (NP) of influenza A/NT/60/68 virus, five Th cell epitopes in B10.S (H-2s), BALB/c (H-2d), CBA (H-2k), and B6 (H-2b) mice have been identified. The specificity of Th cell recognition of epitopes is largely dependent on the H-2 haplotype of the responding mouse strain. However, two out of the five Th epitopes defined could be recognized by mice of more than one haplotype, implying that the primary sequence of protein antigens could also influence the selection of dominant T cell epitopes by the immune system. Immunization of B10.S mice with peptide 260-283 generated strong Th cell response against type A influenza viruses. In the other three strains of mice tested, priming with helper peptides induced a stronger antipeptide than antiviral T cell response. However, the low responsiveness to virus in these mice could be partially overcome by immunization with a mixture of several helper peptides. The Th epitopes are defined by the ability of the peptides to stimulate class II MHC restricted CD4+ T cells to proliferate and to produce IL-2 in vitro. When compared with the known epitopes on NP recognised by class I restricted CD8+ cytotoxic T cells, it appears that Th and cytotoxic T cell epitopes are nonoverlapping. The AMPHI and Motifs methods were employed to analyze the sequence of NP and predict the potential dominant sites in the molecule. The predictions are compared with the experimental data obtained and the implications discussed.     

PRRSV NJ-a株ORF5基因A表位的修饰与糖基化位点的突变对其DNA疫苗免疫效力的影响

为了提高PRRSV ORF5基因的免疫效力,对ORF5基因进行了改造,将CpG序列和通用型辅助性T淋巴细胞表位插入A表位与B表位之间,并对N33与N51位糖基化位点进行了点突变,获得改造的ORF5基因。在此基础上构建了由两个CMV启动子调控的共表达改造的ORF5(MORF5)与ORF6基因的真核表达质粒pcDNA-M5A-6A。经Western-blot与IFA验证真核质粒的体外表达后,免疫6周龄Balb/c小鼠,利用微量中和试验检测免疫后的中和抗体,利用MTT法检测免疫后淋巴细胞的增生情况,并与未改造ORF5基因真核表达质粒pcDNA-5A-6A、弱毒疫苗以及灭活疫苗的免疫效果进行比较。结果表明,pcDNA-M5A-6A不但能够刺激免疫小鼠在较短的时间内产生更高水平的中和抗体,而且可以诱导产生更强烈的T淋巴细胞增殖反应。所构建的共表达PRRSV改造的ORF5基因与ORF6基因的DNA疫苗pcDNA-M5A-6A,能够较好的诱发小鼠产生较高的特异性针对PRRSV的中和抗体和细胞免疫应答,为研究能够更好地防制PRRSV的新型疫苗提供了新的思路。     

Induction of rabies virus-specific T-helper cells by synthetic peptides that carry dominant T-helper cell epitopes of the viral ribonucleoprotein.

The T-helper cell response to the internal proteins of rabies virus was investigated. The rabies virus nucleoprotein was shown to be a major target antigen for T-helper cells that cross-react between rabies and rabies-related viruses. T-helper cells were assayed in vitro by testing virus-induced lymphocytes for lymphokine secretion in response to antigen. Immunodominant T-helper cell epitopes of the viral nucleoprotein were identified in vitro by using synthetic peptides delineated from the amino acid sequence of the nucleoprotein. The response to synthetic peptides were under Ir gene control. Antigenic peptides were tested in vivo for stimulation of rabies virus-specific T-helper cells. Inoculation of mice with peptides bearing immunodominant T-helper cell epitopes resulted in an accelerated and enhanced neutralizing antibody response upon booster immunization with inactivated rabies virus.