Mapping the fine specificity of a cytolytic T cell response to HIV-1 nef protein.

Epitope mapping of a MHC class I-restricted cytotoxic T cell response to nef, a regulatory protein of HIV, was performed with fresh PBMC from HIV-seropositive donors and target cells pulsed with a panel of overlapping peptides of the nef protein. These nef-specific CTL recognized a synthetic peptide of 10 residues derived from a nonamphipathic, highly conserved region of the nef protein in association with the HLA A3.1 molecule. Using human cell transfectants expressing mutations of the A3 molecule, we demonstrated that the amino acid at position 152 of the A3.1 molecule appears to be critical for detection of this response. Thus, rapid analysis of the epitopes of HIV proteins stimulating CTL responses can be achieved using a combination of fresh donor PBMC and target cells pulsed with synthesized peptides.     

Demonstration of two allelic forms of the bovine T cell antigen Bo5 (CD5) and studies of their inheritance

Summary. Two monoclonal antibodies (mAbs), CC17 and IL-A67, which are specific for the bovine equivalent of the CD5 antigen, Bo5, were each found to react with the cells of some animals but not others. The cattle tested were all positive for one or both of the mAbs, but the level of expression on cells expressing both determinants was slightly lower than that on cells expressing either of the determinants on their own. Both mAbs precipitated an antigen of 67kD. However, sequential immunoprecipitation experiments with cells that reacted with both mAbs demonstrated that the determinants are present on two different sets of molecules. These findings suggested that the mAbs recognize two co-dominantly expressed allelic forms of Bo5. This was confirmed in family studies, with groups of full- and half-sibling offspring of sires and dams of defined phenotypes. These experiments also showed that the gene encoding the Bo5 antigen is not linked to the major histo-compatibility complex (MHC). The frequencies of the two alleles, which have been designated Bo5.1 and Bo5.2, in the cattle populations tested were 100% and 0%, respectively, in Bos taurus, and 10% and 90%, respectively, in Bos indicus.     

Demonstration of two allelic forms of the bovine T cell antigen Bo5 (CD5) and studies of their inheritance

Two monoclonal antibodies (mAbs), CC17 and IL-A67, which are specific for the bovine equivalent of the CD5 antigen, Bo5, were each found to react with the cells of some animals but not others. The cattle tested were all positive for one or both of the mAbs, but the level of expression on cells expressing both determinants was slightly lower than that on cells expressing either of the determinants on their own. Both mAbs precipitated an antigen of 67 kD. However, sequential immunoprecipitation experiments with cells that reacted with both mAbs demonstrated that the determinants are present on two different sets of molecules. These findings suggested that the mAbs recognize two co-dominantly expressed allelic forms of Bo5. This was confirmed in family studies, with groups of full- and half-sibling offspring of sires and dams of defined phenotypes. These experiments also showed that the gene encoding the Bo5 antigen is not linked to the major histocompatibility complex (MHC). The frequencies of the two alleles, which have been designated Bo5.1 and Bo5.2, in the cattle populations tested were 100% and 0%, respectively, in Bos taurus, and 10% and 90%, respectively, in Bos indicus.     

A human multi-epitope recombinant vaccinia virus as a universal T cell vaccine candidate against influenza virus

There is a need to develop a universal vaccine against influenza virus infection to avoid developing new formulations of a seasonal vaccine each year. Many of the vaccine strategies for a universal vaccine target strain-conserved influenza virus proteins, such as the matrix, polymerase, and nucleoproteins, rather than the surface hemagglutinin and neuraminidase proteins. In addition, non-disease-causing viral vectors are a popular choice as a delivery system for the influenza virus antigens. As a proof-of-concept, we have designed a novel influenza virus immunogen based on the NP backbone containing human T cell epitopes for M1, NS1, NP, PB1 and PA proteins (referred as NPmix) as well as a construct containing the conserved regions of influenza virus neuraminidase (N-terminal) and hemagglutinin (C-terminal) (referred as NA-HA). DNA vectors and vaccinia virus recombinants expressing NPmix (WR-NP) or both NPmix plus NA-HA (WR-flu) in the cytosol were tested in a heterologous DNA-prime/vaccinia virus-boost vaccine regimen in mice. We observed an increase in the number of influenza virus-specific IFNγ-secreting splenocytes, composed of populations marked by CD4(+) and CD8(+) T cells producing IFNγ or TNFα. Upon challenge with influenza virus, the vaccinated mice exhibited decreased viral load in the lungs and a delay in mortality. These findings suggest that DNA prime/poxvirus boost with human multi-epitope recombinant influenza virus proteins is a valid approach for a general T-cell vaccine to protect against influenza virus infection.     

Characterization of two allelic forms of Neurospora crassa laccase. Amino- and carboxyl-terminal processing of a precursor

The complete structures of the laccase genes isolated from two different Neurospora crassa wild-type strains are described. The genes were cloned by screening partial genomic DNA libraries with a nick-translated laccase-specified 1.36-kilobase SalI fragment (Germann, U. A., and Lerch, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8854-8858) as a hybridization probe. Nucleotide sequence analysis revealed the presence of two different allelic forms. They conform to the same structural organization, but show an overall divergence of 5.3% which is mainly the result of point mutations in the nontranslated regions. The coding parts are interrupted by a short intron. The encoded proteins differ in 12 out of 619 amino acid residues. A comparison of the primary structure deduced from the nucleotide sequence of the gene with a protein chemical analysis of the two terminal cyanogen bromide fragments of extracellular N. crassa laccase revealed that the enzyme is synthesized as a precursor. The precursor protein exceeds the mature protein by 49 amino acids at its amino terminus and by 13 amino acids at its carboxyl terminus, thus indicating a complex maturation pathway. The possible involvement of amino-terminal processing in secretion and of carboxyl-terminal processing in activation of the enzyme is discussed.     

T cell function during fatal and self-limiting malarial infections of mice.

Delayed-type hypersensitivity (DTH) to sheep erythrocytes (SRBC) was found to be depressed during fatal Plasmodium berghei and self-limiting P. yoelii infections of mice. By testing mice presensitized to SRBC before P. berghei infection, and by transfer of cells sensitized in uninfected mice into P. yoelii-infected recipients, the immunological lesion was found to be at the level of DTH expression, rather than at the level of T cell sensitization. That acute inflammatory responsiveness is impaired during malarial infection was confirmed by testing this response to local injection of LPS in P. yoelii-infected mice. The results suggested that depressed DTH responsiveness in malarious mice is not a valid indication of impaired T cell function.     

Unusually diverse T cell response to a repeating tripeptide epitope.

The immune system utilizes a diverse T cell repertoire for the recognition of foreign antigens in the context of self MHC gene products. We have examined the potential diversity of the T cell response directed to a immunodominant repeating tripeptide epitope (EYA)5. This peptide represents one of the two T cell epitopes on the synthetic alpha-helical polypeptide antigen Poly 18, Poly EYK(EYA)5 in H-2d mice and does not require antigen processing prior to presentation to Poly 18-specific T cell hybridomas. The T cell response directed to the repeating tripeptide epitope (EYA)5 is extremely heterogenous even though the epitope has a relatively simple amino acid sequence. We have analyzed the fine specificity of 21 randomly chosen Poly 18-reactive, (EYA)5-specific and H-2d-restricted T cell hybridomas derived from H-2d, H-2bxd, and H-2b----H-2bxd Poly 18-responding mice to determine the number of unique antigen reactivity patterns represented by this T cell population. We used alanine- and/or lysine-substituted (EYA)5 peptides and a panel of haplotype-varied splenocytes and observed a great deal of microheterogeneity in response. We find that 13 of the 21 hybridomas have a distinct fine antigen specificity and T cell receptors. The binding of (EYA)5 to the antigen-binding groove of I-Ad appears to generate a highly diversified T cell response. Therefore, (EYA)5-I-Ad complex allows the activation of unrelated T cell clonotypes with the same overall antigen specificity and MHC restriction, but with distinct microheterogeneity in response and receptor usage.     

Collaborative study to assess the suitability of a candidate International Standard for yellow fever vaccine

Yellow fever vaccines are routinely assayed by plaque assay. However, the results of these assays are then converted into mouse LD(50) using correlations/conversion factors which, in many cases, were established many years ago. The minimum required potency in WHO Recommendations is 10(3) LD(50)/dose. Thirteen participants from 8 countries participated in a collaborative study whose aim was to assess the suitability of two candidate preparations to serve as an International Standard for yellow fever vaccine. In addition, the study investigated the relationship between the mouse LD(50) test and plaque forming units with a view to updating the WHO recommendations. Plaque assays were more reproducible than mouse assays, as expected. Differences in sensitivities of plaque assays were observed between laboratories but these differences appear to be consistent within a laboratory for all samples and the expression of potency relative to the candidate standard vaccine improved the reproducibility of assays between laboratories. However, the use of potencies had little effect on the between laboratory variability in mouse LD(50) assays. There appears to be a consistent relationship between overall mean LD(50) and plaques titre for all study preparations other than sample E. The slope of the correlation curve is >1 and it would appear that 10(3) LD(50) is approximately equivalent to 10(4) plaque forming units (PFU), based on the overall means of all laboratory results. The First International Standard for yellow fever vaccine, NIBSC Code 99/616, has been established as the First International Standard for yellow fever vaccine by the Expert Committee of Biological Standards of the World Health Organisation. The International Standard has been arbitrarily assigned a potency of 10(4.5) International Units (IU) per ampoule. Manufacturers and National Control Laboratories are including the First International Standard for yellow fever vaccine in routine assays so that the minimum potency in IU of vaccines released for use and which meet the current minimum potency of 10(3) LD(50) in mouse assays, can be determined. These data will be analysed before a review of the WHO requirements, including the minimum potency per dose, is undertaken.     

Dramatic efficacy improvement of a DC-based vaccine against AML by CD25 T cell depletion allowing the induction of a long-lasting T cell response

Dendritic cell (DC)-based vaccination is a promising approach to enhance anti-tumor immunity that could be considered for acute myeloid leukemia (AML) patients with high-risk of relapse. Our purpose was to study the efficiency and to optimize the immunogenicity of a DC-based vaccine in a preclinical AML murine model. In this report, C57BL6 mice were vaccinated with DC pulsed with peptides eluted (EP) from the syngeneic C1498 myelomonocytic leukemic cell line in a prophylactic setting. In this model, a natural antileukemic immunity mediated by NK cells was observed in the control unloaded DC-vaccinated group. On the other hand, we showed that the cytotoxic antileukemic immune response induced by vaccination with eluted peptides pulsed-DC (DC/EP), in vitro and in vivo, was mainly mediated by CD4⁺ T cells. Treatment with anti-CD25 antibody to deplete CD4⁺ CD25⁺ regulatory T cells before DC-vaccination dramatically improved the antileukemic immune response induced by immunization, and allowed the development of long-lasting immune responses that were tumor protective after a re-challenge with leukemic cells. Our results suggest that this approach could be successful against weakly immunogenic tumors such as AML, and could be translated in human.     

Interdependence of CD4+ T cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development

We studied immunity to the blood stage of the rodent malaria, Plasmodium vinckei vinckei, which is uniformly lethal to mice. BALB/c mice develop solid immunity after two infections and drug cure. The following experiments define the basis of this immunity. Transfer of pooled serum from such immune mice renders very limited protection to BALB/c mice and no protection to athymic nu/nu mice. Moreover, B cell-deficient C3H/HeN mice develop immunity to P. vinckei reinfection in the same manner as immunologically intact mice, an observation made earlier. In vivo depletion of CD4+ T cells in immune mice abrogates their immunity. This loss of immunity could be reversed through reconstitution of in vivo CD4-depleted mice with fractionated B-, CD8-, CD4+ immune spleen cells; however, adoptive transfer of fractionated CD4+ T cells from immune spleen into naive BALB/c or histocompatible BALB/c nude mice does not render recipients immune. In vivo depletion of CD8+ T cells did not influence the parasitemia in nonimmune or immune mice. Splenectomy of immune mice completely reverses their immunity. Repletion of splenectomized mice with their own spleen cells does not reconstitute their immunity. We conclude that some feature of the malaria-modified spleen acts in concert with the effector/inducer function of CD4+ T cells to provide protection from P. vinckei. To be consistent with this finding, a malaria vaccine may require a combination of malaria Ag to induce immune CD4+ T cells and an adjuvant or other vaccine vehicle to alter the spleen.     

Pertussis toxin prevents the induction of peripheral T cell anergy and enhances the T cell response to an encephalitogenic peptide of myelin basic protein.

In a murine model of T cell-mediated autoimmune disease, experimental autoimmune encephalitis (EAE), 80% of all encephalitogenic T cell clones in H-2u mice use the V beta 8.2 TCR element. To induce EAE in susceptible strains of mice either heat-killed Bordetella pertussis organisms or Bordetella pertussis toxin (PT) must be injected in addition to Ag in CFA. We investigated the mechanisms by which PT facilitates the induction of EAE. Our data show, that PT interferes with the induction of Ag-induced peripheral T cell anergy. Furthermore it has a specific adjuvanticity for the autoantigen pAc1-11 in vivo and acts as a selective mitogen in vitro. We also tested the hypothesis that PT is a bacterial superantigen that specifically expands the V beta 8.2+ subset of T cells, thereby expanding the encephalitogenic T cell clones that are contained in this subset, so that the number of autoreactive T cells is brought over a critical threshold, necessary to induce autoimmune disease. Our data show that PT is not a superantigen. Staphylococcal enterotoxin B, a V beta 8.2-specific superantigen, does not enhance the immune response to the encephalitogenic peptide.     

Identification of immunodominant Th1-type T cell epitopes from Schistosoma japonicum 28 kDa glutathione-S-transferase, a vaccine candidate

Th1-type cytokines produced by the stimulation of Th 1-type epitopes derived from defined schistosome-associated antigens are correlated with the development of resistance to the parasite infection. Schistosoma mansoni 28 kDa glutathione-S-transferase （Sm28GST）, a major detoxification enzyme, has been recognized as a vaccine candidate and a phase II clinical trial has been carried out. Sheep immunized with recombinant Schistosoma japonicum 28GST （Sj28GST） have shown immune protection against the parasite infection. In the present study, six candidate peptides （P1, P2, P3, P4, P7 and P8） from Sj28GST were predicted, using software, to be T cell epitopes, and peptides P5 and P6 were designed by extending five amino acids at the N-terminal and C-terminal of P1, respectively. The peptide 190-211 aa in Sj28GST corresponding to the Th1-type epitope （190-211 aa） identified from Sm28GST was selected and named P9. The nine candidate peptides were synthesized or produced as the fusion protein with thioredoxin in the pET32c（＋）/BL21（DE3） system. Their capacity to induce a Th1-type response in vitro was measured using lymphocyte proliferation, cytokine detection experiments and flow cytometry. The results showed that P6 （73-86 aa） generated the strongest stimulation effect on T cells among the nine candidate peptides, and drove the highest level of IFN-γ, and IL-2. Therefore, P6 is a functional Thl-type T cell epitope that is different from that in Sm28GST, and will be useful for the development of effective vaccines which can trigger acquired immunity against S. japonicum. Moreover, our strategy of identifying the Thl-type epitope by a combination of software prediction and experimental confirmation provides a convenient and cost-saving alternative approach to previous methods.     

Prediction of B cell and T cell epitopes of DBLalpha domain in Plasmodium falciparum malaria vaccine candidate var gene

P. falciparum encodes PfEMP-1 which is important in pathogenicity and virulence. We have analyzed the structure of the Duffy binding like (DBLalpha) domain of var genes and predicted the antigenic sites and T and B cell epitopes which present a highly variable picture with major implications in immune interactions and vaccine design.     

Identifying and modulating disulfide formation in the biopharmaceutical production of a recombinant protein vaccine candidate

Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.     

Synthesis of a Streptococcus pyogenes vaccine candidate based on the M protein PL1 epitope

Group A streptococcus is a Gram-positive bacteria that causes a range of infectious diseases. Targeting the bacteria, a new self-adjuvanting vaccine candidate, incorporating a carbohydrate carrier and an amino acid-based adjuvant, was synthesised utilising carbohydrate chemistry and solid-phase peptide synthesis procedures. Characterisation of the candidate was achieved using reverse-phase HPLC and electrospray ionisation mass spectrometry. The successful synthesis and characterisation of the vaccine candidate may contribute to the discovery of a therapeutically and clinically viable vaccine against group A streptococcus.     

Characterization of the immune response to a secondary encephalitogenic epitope of basic protein in Lewis rats. I. T cell receptor peptide regulation of T cell clones expressing cross-reactive V beta genes.

In Lewis rats, immunization with myelin basic protein induces two distinct encephalitogenic T cell populations, those responding to the immunodominant 72-89 epitope and those specific for a secondary epitope including residues 87-99. The 72-89 specific T cells were I-A restricted and preferentially expressed V beta 8.2 in their TCR. To determine the fine specificity, MHC restriction, and TCR V beta gene use in T cells reactive to the secondary epitope, we characterized 23 T cell clones from the lymph nodes (LN) and spinal cords (SC) of rats immunized with either whole basic protein or synthetic peptides S85-99 and S87-99 that were found to be functionally similar. The S85-99/S87-99 specific clones from LN and SC were all encephalitogenic despite differences in recognition of intact basic protein and class II MHC restriction. Unlike LN clones that overexpressed V beta 8 (46%+) and V beta 6 (31%+), however, SC clones were strongly biased (86%+) in their expression of V beta 6. This V gene bias raised the possibility of TCR peptide therapy using V beta 6 peptides. The V beta 6 sequence was similar to V beta 8.2 in the CDR2 region, and the corresponding peptides from this region were found to be cross-reactive in vivo. Moreover, both peptides were effective in the treatment of EAE induced with either S85-99, biased in V beta 6+ and V beta 8+ T cells, or guinea pig basic protein, biased only in V beta 8+ T cells. These data demonstrate the presence of common immunogenic epitopes among subsets of TCR V region gene families that possess important regulatory activity on effector T cell function.     

Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene.

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.