Specificity of the T-cell responses in covalently linked peptides each comprising of a T helper epitope

The selection of T-cell specificities in chimeric peptides comprising of two T helper epitopes (288-302 and 240-252) from the fusion protein of measles virus was investigated. The resulting chimeric peptides (288-P-240 and 240-P-288) were shown to be immunogenic by inducing proliferative responses in both B10.s and C57BL/6 strains of mice. In B10.s mice immunization with the chimeric peptides resulted in proliferative T-cell responses only to the constituent 288-302 peptide, whereas in C57BL/6 mice no proliferative responses to the constituent 288-302 or 240-252 peptides were detected. In vivo competition studies between the 288-302 and 240-252 peptides for binding to the I-As molecule have shown that the peptide 288-302 was dominant in B10.s mice and competed with the non-dominant 240-252 peptide for the induction of an in vivo response. The absence of any proliferative T-cell response to the constituent 288-302 and 240-252 peptides after immunization of C57BL/6 mice with the 288-P-240 or 240-P-288 chimeric peptides, suggests that the dominant T-cell responses might have shifted to newly formed T cell epitope(s) as a result of the covalent linkage of the two peptides. In conclusion, these results indicate that the selection of Th epitopes within chimeric peptides is dependent not only on the amino acid composition of the epitope but also on the context of the epitope within the chimera and the haplotype of the mouse strain used.     

Cross-reactive and group-specific immune responses to a neutralizing epitope of the human respiratory syncytial virus fusion protein

Immune responses to a synthetic peptide corresponding to amino-acids 205-225 of the fusion protein from group B respiratory syncytial (RS) virus, were studied in mice and rabbits, and compared to a similar peptide from group A RS virus. Peptide 205-225 (B) was recognized by monoclonal antibody RS-348, and was immunogenic in both mice and rabbits, as was peptide 205-225 from the fusion protein of a group A strain. Peptide 205-225 (B) induced a proliferative T-cell response, demonstrating the existence of a T-cell epitope in this region of the fusion protein of group B viruses. Both peptides were able to induce a T-cell cross-reactive proliferation when mice were primed with either the homologous or the heterologous peptide. ELISA were performed using synthetic peptides or whole virus (from group A and B) as antigens. Mice anti-peptide sera recognized both homologous and heterologous peptides. A similar pattern was observed with RS virus strains. In indirect immunofluorescence assays, both anti-peptide rabbit sera recognized human nasal epithelial cells infected with A or B strains of RS virus. In contrast, while anti-peptide 205-225 rabbit serum from group A neutralized group A and B strains of RS virus, anti-peptide 205-225 rabbit serum from group B was unable to neutralize a group A virus, although it neutralized a group B strain. These results are similar to the immune response observed in children following primary RS virus infection.     

Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein

Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.     

Protective activity of Borrelia duttonii-specific immunoglobulin subclasses in mice

To analyse the immune response of mice to Borrelia duttonii infection, BALB/c mice were inoculated intraperitoneally with B. duttonii strain 406K, and the titres of B. duttonii-specific immunoglobulins - IgM, IgG1, IgG2a, IgG2b and IgG3 - were determined by ELISA. IgM antibodies appeared first, followed by IgG2a and IgG3, and then IgG1 and IgG2b. The protective activity of individual classes and subclasses of B. duttonii-specific immunoglobulins was then determined by passive immunisation of BALB/c mice with immunoglobulin preparations purified by affinity chromatography. The mice were then challenged by intraperitoneal inoculation of B. duttonii. The study demonstrated that B. duttonii-specific IgM and IgG3 protected against the development of spirochaetaemia and death after borrelial infection, whereas B. duttonii-specific IgG1, IgG2a and IgG2b had low protective activities.     

A nonimmunodominant nucleoprotein-derived peptide is presented by influenza A virus-infected H-2b cells

Influenza A virus-infected H-2b mice mount a CTL response directed against the nucleoprotein (NP) 366-374 but not against the NP 55-63 peptide, although both peptides fulfill the prerequisites for having high binding affinity toward the Db molecule. Two hypotheses have been proposed to explain the inability of B6 mice to respond to the NP 55-63 peptide: 1) B6 mice are tolerant to the NP 55-63 peptide and 2) NP 55-63 peptide is not naturally processed by H-2b cells. Our results show that 1) B6 mice possess a NP 55-63-specific CTL repertoire because their immunization with the NP 55-63 peptide itself recruits specific CTL; 2) NP 55-63 peptide is naturally processed by the virus-infected H-2b cells but its efficient presentation by the Db molecule requires higher amounts of NP than presentation of the NP 366-374 peptide; 3) NP 55-63 peptide is naturally presented in virus infected B6 mice, however, the quantity of Db/NP 55-63 complexes at the cell surface is sufficient to tolerize but not to recruit and stimulate specific CTL; and 4) NP 55-63 peptide binds to the Db molecule with a lower affinity than NP 366-374 does and this difference could explain the inefficient presentation of the NP 55-63 peptide by B6 cells. The involvement of the self-protein-derived nonimmunodominant peptides in self-tolerance and the possibility of using nonimmunodominant peptides of viral proteins for peptide vaccination are discussed.     

Inactivation of T cell receptor peptide-specific CD4 regulatory T cells induces chronic experimental autoimmune encephalomyelitis (EAE)

T cell receptor (TCR)-recognizing regulatory cells, induced after vaccination with self-reactive T cells or TCR peptides, have been shown to prevent autoimmunity. We have asked whether this regulation is involved in the maintenance of peripheral tolerance to myelin basic protein (MBP) in an autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). Antigen-induced EAE in (SJL x B10.PL)F1 mice is transient in that most animals recover permanently from the disease. Most of the initial encephalitogenic T cells recognize MBP Ac1-9 and predominantly use the TCR V beta 8.2 gene segment. In mice recovering from MBP-induced EAE, regulatory CD4+ T cells (Treg) specific for a single immunodominant TCR peptide B5 (76-101) from framework region 3 of the V beta 8.2 chain, become primed. We have earlier shown that cloned B5-reactive Treg can specifically downregulate responses to Ac1-9 and also protect mice from EAE. These CD4 Treg clones predominantly use the TCR V beta 14 or V beta 3 gene segments. Here we have directly tested whether deletion/blocking of the Treg from the peripheral repertoire affects the spontaneous recovery from EAE. Treatment of F1 mice with appropriate V beta-specific monoclonal antibodies resulted in an increase in the severity and duration of the disease; even relapses were seen in one-third to one-half of the Treg-deleted mice. Interestingly, chronic disease in treated mice appears to be due to the presence of Ac1-9-specific T cells. Thus, once self-tolerance to MBP is broken by immunization with the antigen in strong adjuvant, TCR peptide-specific CD4 Treg cells participate in reestablishing peripheral tolerance. Thus, a failure to generate Treg may be implicated in chronic autoimmune conditions.     

A peptide mimic of a protective epitope of respiratory syncytial virus selected from a combinatorial library induces virus-neutralizing antibodies and reduces viral load in vivo

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 x 10(9) and 4.93 x 10(9) M(-1) for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.     

Hemodynamic effects of spinal anesthesia in the elderly: single dose versus titration through a catheter

Protective immunity against infection with Mycobacterium tuberculosis is imparted by T cells rather than antibodies, but B cells can play a role as antigen-presenting cells and in granuloma formation. We re-evaluated the role of B cells in the course of tuberculous infection in mu-chain knock-out (Ig-) mice. Surprisingly, the organs of M. tuberculosis-infected Ig- mice were found to have three- to eight-fold elevated counts of viable bacilli compared with normal littermates at 3-6 weeks post-infection. Splenic interferon-gamma responses to whole antigen were unimpaired, whilst proliferation to certain mycobacterial peptides was found to be diminished. However, bacille Calmette-Guérin (BCG) vaccination significantly reduced the infection in Ig- mice. The mechanisms by which B cells can influence primary tuberculous infection need further study.     

Characterization of a peptide analog of a determinant of type II collagen that suppresses collagen-induced arthritis

Immunization of susceptible strains of mice with type II collagen (CII) elicits an autoimmune arthritis known as collagen-induced arthritis (CIA). One analogue peptide of the immunodominant T cell determinant, A9 (CII245-270 (I260-->A, A261-->B, F263-->N)), was previously shown to induce a profound suppression of CIA when coadministered at the time of immunization with CII. In the present study, A9 peptide was administered i.p., orally, intranasally, or i.v. 2 to 4 wk following CII immunization. We found that arthritis was significantly suppressed even when A9 was administered after disease was induced. To determine the mechanism of action of A9, cytokine responses to A9 and wild-type peptide A2 by CII-sensitized spleen cells were compared. An increase in IL-4 and IL-10, but not in IFN-gamma, was found in A9 culture supernatants. Additionally, cells obtained from A9-immunized mice produced higher amounts of IL-4 and IL-10 when cultured with CII compared with cells obtained from mice immunized with A2, which produced predominantly IFN-gamma. Suppression of arthritis could be transferred to naive mice using A9-immune splenocytes. Lastly, phosphorylation of TCRzeta was not altered in the immunoprecipitates from the lysates of cells exposed to analogue peptides (A9 and A10) together with wild-type A2 in a T cell line and two I-Aq-restricted, CII-specific T hybridomas. We conclude that analogue peptide A9 is effective in suppressing established CIA by inducing T cells to produce a Th2 cytokine pattern in response to CII.     

Intravital videomicroscopic evidence for regulation of metastasis by the hepatic microvasculature: effects of interleukin-1alpha on metastasis and the location of B16F1 melanoma cell arrest

There have been few reported visual observations of metastatic cancer cell arrest in vivo. To seek evidence that inducible vascular adhesive properties can regulate hepatic metastasis, groups of 9-14 c57bl/6 mice were given 1.5 microg of interleukin-1alpha (IL-1alpha) 4 h before the injection of 3 x 10(5) B16F1 melanoma cells into a mesenteric vein. After 7 days, these mice had an 11-22-fold greater hepatic tumor burden than controls given i.p. saline. In both groups, small metastases were seen in the portal tract region. Twice as many 125I-labeled UdR-labeled B16F1 cells were detected in the livers of IL-1alpha-treated animals 5 min after injection, and 7 times as many were found after 24 h. Intravital videomicroscopy showed marked differences in the arrest pattern of the B16F1 cells between controls and IL-1alpha-treated mice. In controls, arrest occurred at a median distance of 32 microm beyond the sinusoidal inlet, where the median sinusoidal diameter was 16 microm. However, in IL-1alpha-treated mice, arrest occurred in the presinusoidal portal vein branches, which had a median diameter of 34 microm. Maximum observed tumor cell velocities were 2-fold less in the IL-1alpha-treated mice, although there was no significant difference in the flow rate of RBCs. To look for effects on the adhesive properties of the hepatic microvasculature, 5 x 10(4) B16F1 cells were incubated for 15 min on 5-microm sections of liver from control and IL-1alpha-treated mice. Three-fold more cells adhered to sections of liver from IL-1alpha-treated mice. This phenomenon was blocked by GRGDS peptides and by antibodies to E-selectin, ICAM-1, VCAM-1, and the alpha v integrin subunit. We postulate that pretreatment of mice with IL-1alpha alters a number of adhesive interactions between B16F1 cells and the hepatic microvasculature, contributing to the site of arrest and to the subsequent fate of the arrested cells.     

Short report: identification of B-cell epitopes in the serine-rich Entamoeba histolytica protein

The serine-rich Entamoeba histolytica protein (SREHP) has been shown to be a protective antigen in animal models of amebic liver abscess when delivered by either parenteral or oral routes of immunization, and antibodies to SREHP can prevent amebic liver abscess in severe combined immunodeficient mice. To identify B cell epitopes of the SREHP molecule that could serve as the basis for a peptide-based vaccine, we synthesized overlapping peptides spanning the amino acid sequence of SREHP, and looked at the reactivity of serum samples from five individuals with amebic liver abscess to the overlapping peptides. We found that most of the epitopes recognized by serum samples from patients with amebic liver abscess map to the hydrophilic dodecapeptide or octapeptide repeats of SREHP, but there was no universal epitope recognized by all five serum samples. In addition, we show that synthetic peptides that include the epitopes of SREHP recognized in the mapping study are immunogenic in animals and can generate antibodies that recognize SREHP.     

Humoral immunity to Borrelia burgdorferi N40 decorin binding proteins during infection of laboratory mice

A Borrelia burgdorferi N40 genomic expression library was screened with serum from actively infected mice to identify gene products that elicit protective immunity. A clone that contained a putative bicistronic operon containing two genes that encoded 20- and 22-kDa lipoproteins was identified and sequenced. These genes showed homology with the genes encoding decorin binding proteins DbpB and DbpA, respectively, of B. burgdorferi 297 and B31. N40-dbpA DNA hybridized with B. burgdorferi N40 DNA on a single 48-kb linear plasmid. Homologous genes could be amplified under various degrees of stringency by PCR or hybridized by Southern blotting from B. burgdorferi sensu stricto N40 and B31, and from B. burgdorferi sensu lato PBi and 25015, but not PKo. Recombinant N40-DbpB and N40-DbpA were reactive with antibody in serum from infected mice, and serum was more reactive against N40-DbpA than against B. burgdorferi N40 recombinant P39, OspC, or OspA. Sera from mice infected with B. burgdorferi sensu lato strains PKo and PBi were weakly reactive against N40-DbpB and N40-DbpA, and sera from mice infected with 25015 were moderately reactive, compared to sera from mice infected with B. burgdorferi N40. Hyperimmunization of mice with N40-DbpA, but not N40-DbpB, induced protective immunity against syringe challenge with cultured B. burgdorferi N40. DbpA may therefore be one of the antigens responsible for eliciting protective antibody known to exist in serum from infected mice. DNA amplification and serology suggest that DbpB and DbpA are likely to have homologs throughout the B. burgdorferi sensu lato family, but they are likely to be heterogeneous.     

Epitope polarity and adjuvants influence the fine specificity of the humoral response against Semliki Forest virus specific peptide vaccines

The humoral response to synthetic peptide vaccines against Semliki Forest virus (SFV) in H-2d BALB/c mice was investigated with the enzyme linked immunosorbent assay and the pepscan technique. The peptide vaccines consisted of amino acid sequences 240-255 (B) and 137-151 (T) of the E2 membrane protein of SFV colinearly synthesized in either orientation T-B or B-T. Sequence B contains an epitope inducing humoral immunity to lethal SFV infection and sequence T contains a H-2d restricted T-helper cell epitope. With sera from mice immunized subcutaneously with peptide T-B, and Quil A as adjuvant, two immunodominant B-cell epitopes were identified, FVPRAD, at position 240-246 and PHYGKEI, at position 145-151. However, with peptide B-T and Quil A as adjuvant for immunization the epitope PHYGKEI was clearly immunodominant, but antibodies elicited against this epitope were not reactive with SFV-infected L cells in contrast to the antibodies elicited by epitope FVPRAD. An additional epitope EPARKGKVH, at position 247-255, was identified with sera from mice immunized subcutaneously with either peptide T-B or B-T and Montanide ISA 740 as an adjuvant. Monoclonal antibodies selected for reactivity with SFV-infected L cells did bind also to epitope FVPRAD. Interestingly, this epitope could induce antibodies cross-reactive with a synthetic peptide derived from macrophage migration inhibitory factor that shares amino acid residues VPRA at position 9-12 with the protective B-cell epitope FVPRAD. The present study shows clearly that the fine specificity of the humoral response against peptide vaccines is differentially influenced by both adjuvant and epitope polarity which may affect vaccine efficacy. Further, the study reminds us that potentially autoimmune antibodies could be induced by vaccines.     

Major histocompatibility complex class II expression and parathyroid autoantibodies in primary hyperparathyroidism

We evaluated whether kinins exert a protective action against the development of two-kidney, one clip (2K1C) hypertension, a model characterized by an activated renin-angiotensin system in the ischemic kidney and increased expression of the bradykinin (BK) B2 receptor in the contralateral kidney. BK B2-receptor knockout (B2-/-), wild-type (B2+/+), and heterozygous (B2+/-) mice underwent clipping of the left renal artery, with the other kidney remaining untouched. Basal systolic blood pressure (SBP, via tail-cuff plethysmography) was higher in B2-/- mice than in B2+/- or B2+/+ mice (121+/-2 versus 113+/-2 and 109+/-1 mm Hg; P<0.05 for both comparisons). SBP did not change from basal values after sham operation, but it increased in mice that underwent clipping. The increase in SBP was greater in 2K1C B2-/- mice than in B2+/- or B2+/+ mice (28+/-2 versus 14+/-2 and 14+/-2 mm Hg, respectively, at 2 weeks; P<0.05 for both comparisons). Blockade of the BK B2 receptor by Icatibant enhanced the pressure response to clipping in B2+/+ mice (29+/-2 mm Hg at 2 weeks). Intra-arterial mean blood pressure (MBP) was higher in 2K1C than in respective sham-operated mice, with the MBP difference being higher in B2-/- mice (32 and 38 mm Hg, at 2 and 4 weeks, respectively), and higher in B2+/+ mice given Icatibant (30 and 32 mm Hg) than in B2+/+ mice without Icatibant (17 and 18 mm Hg). At 4 weeks, acute injection of an angiotensin type 1 receptor antagonist normalized the MBP of 2K1C hypertensive mice. A tachycardic response was observed 1 week after clipping in B2-/- and B2+/- mice, but this effect was delayed in B2+/+ mice. However, the HR response to clipping in B2+/+ mice was enhanced by Icatibant. Within each strain, heart weight to body weight ratio was greater in 2K1C hypertensive mice than in sham-operated control animals (B2-/-: 5.7+/-0.1 versus 5.2+/-0.1; B2+/+: 5.1+/-0.1 versus 4.5+/-0.1; P<0.01 for both comparisons). The clipped kidney weight to nonclipped kidney weight ratio was consistently reduced in mice with 2K1C hypertension. Our results indicate that kinins acting on the BK B2 receptor exert a protective action against excessive blood pressure elevation during early phases of 2K1C hypertension.     

Immune defense and eosinophilia in congenitally IgE-deficient SJA/9 mice infected with Angiostrongylus costaricensis

The roles of IgE in protective immunity and eosinophilia in Angiostrongylus costaricensis infection were examined by comparing IgE-deficient SJA/9 mice and IgE-producing SJL/J mice. In primary infection, mean total IgE levels increased to a maximum of 390 ng/ml, which was more than 10 times greater than the 29 ng/ml measured preinfection in SJL/J mice but less than the 10 ng/ml found in SJA/9 mice throughout the experiment. Immune defense as determined by recovery of adult worms and eosinophilia were similar in SJL/J and SJA/9 mice. Protective immunity was induced by infection with A. costaricensis followed by treatment with levamisole for 4-6 days postinfection. After the challenge infection, the numbers of adult worms and eosinophils in SJA/9 mice were not significantly different from those in SJL/J mice. Anti-A. costaricensis IgE antibody was not detected in either strain of mice during the experiment. These results indicate that A. costaricensis infection induced the production of IgE not specific for parasite antigens in IgE-producing mice. Potentiated nonspecific IgE played no significant role in immune defense and eosinophilia.     

Mapping of conformational B cell epitopes within alpha-helical coiled coil proteins

An approach to mapping antigenic B cell epitopes within alpha-helical coiled coil proteins has been developed and applied to two proteins: Streptococcal M protein and C. elegans paramyosin protein UNC-15. Overlapping peptides derived from an alpha-helical coiled coil conformational epitope were embedded between helical flanking peptides derived from the completely unrelated GCN4 leucine zipper peptide. The resulting chimeric peptides exhibited helical propensity. Chimeric peptides were tested for antigenicity (recognition by antibody) or immunogenicity (production of appropriate antibody response). A conformational epitope within the Streptococcal M protein recognised by three mAbs spanned 12 residues. Analysis of chimeric peptides based on C. elegans UNC-15 has enabled fine mapping of the minimal B cell epitope recognised by monoclonal antibody NE1-6B2 to seven non-contiguous residues (spanning 15 residues); the footprint of contact residues involved in antibody recognition being restricted to the hydrophilic face of the helix and covering five helical turns. This chimeric peptide epitope when coupled to diphtheria toxoid was highly immunogenic in mice and antisera recognised the conformationally dependent native peptide epitope. This approach has the potential to map conformational epitopes and design minimal epitopes for use as vaccine candidates.     

Intraductal papillary mucinous tumors of the pancreas: imaging studies and treatment strategies

Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70-peptide mixture and challenged with LCMV. Virus titers were reduced 10-100-fold in these mice compared to control mice. Immunization with the hsp70-peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells.     

Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4(+) human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4(+) T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1-20. DR3.Ab0 mice, immunized with bacillus Calmette-Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1-20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette-Guérin but not to p1-20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171-200, 311-340, and 411-440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51-70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1-20 and p51-70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.     

A vaccine and monoclonal antibodies that enhance mouse resistance to Candida albicans vaginal infection

We previously reported that a vaccine composed of liposome-mannan complexes of Candida albicans (L-mann) stimulates mice to produce protective antibodies against disseminated candidiasis. An immunoglobulin M (IgM) monoclonal antibody (MAb), B6.1, specific for a beta-1,2-mannotriose in the complexes protects against the disease, whereas MAb B6 does not. In the present study, the vaccine and MAbs B6.1 and B6 were tested for the ability to protect against Candida vaginal infection, established by intravaginal (i.vg.) inoculation of yeast cells in mice maintained in pseudoestrus. Fungal CFU in each vagina was determined to assess the severity of infection. Mice vaccinated before infection developed about 62% fewer vaginal CFU than nonimmunized controls. Naive mice that received polyclonal antiserum (from vaccinated mice) i.vg. before infection had 60% fewer CFU than controls. The serum protective factor was stable at 56 degreesC, but C. albicans cells absorbed this factor. Mice given MAb B6.1 i.vg. after infection was established had fewer Candida CFU in vaginal tissue than control mice given buffer instead of antibody. MAbs B6.1 and B6 given intraperitoneally before infection protected mice, but MAbs preabsorbed with yeast cells did not. MAb B6.1 also protected against C. tropicalis vaginal infection, but MAb B6 did not. The protective activities of MAbs B6.1 and B6 appeared to be specific because an irrelevant IgM carbohydrate-specific MAb and an irrelevant IgG protein-specific MAb were not protective; also, MAb B6.1 did not affect development of vaginal chlamydial infection. These studies show that an appropriate antibody response, or administration of protective antibodies, can help the host to resist Candida vaginal infection.     

Cross-priming of CTL responses in vivo does not require antigenic peptides in the endoplasmic reticulum of immunizing cells

It has been proposed that the cross-priming of CTL responses in vivo involves the transfer to host APCs of heat shock protein glycoprotein 96-chaperoned antigenic peptides released from the endoplasmic reticulum (ER) of dying or infected cells. We have tested this possibility directly using TAP-deficient cell lines lacking antigenic ER peptides derived from two model Ags, the human adenovirus type 5 early regions E1A and E1B. Although both proteins were well expressed, the cells were not recognized by E1A- or E1B-specific CTLs unless the relevant epitope was either provided exogenously as a synthetic peptide or targeted to the ER in a TAP-independent fashion. Despite the absence of these ER peptides, the TAP1-/- cells were able to efficiently cross-prime E1A- and E1B-specific CTLs following immunization of syngeneic mice. These results indicate that, although purified peptide/glycoprotein 96 complexes are potent immunogens, the mechanism of CTL cross-priming in vivo does not depend upon antigenic peptides in the ER of immunizing cells.