Type D SRV-2 virus-specific CD8+ and CD4- CD8- T cells that regulate virus-induced T cell proliferation in Celebes macaques

These studies defined SRV-2 envelope peptides 96-102, 127-152, and 233-249 as T cell epitopes that induce significant T cell proliferation. Peripheral blood lymphocytes of Celebes macaques (Macaca nigra) exposed to SRV-2 and currently virus^- antibody⁺, cultured with SRV-2 virus show strongly suppressed T cell responses and have two immunoregulatory T cell populations.     

Synthetic immunogens constructed from T-cell and B-cell stimulating peptides (T:B chimeras): preferential stimulation of unique T- and B-cell specificities is influenced by immunogen configuration.

In our effort to develop synthetic immunogens as vaccines, we have focused on the combination of a known T-cell stimulating peptide with putative B-cell stimulating peptide epitopes derived from the sequences of respiratory syncytial (RS) virus proteins. The T-cell stimulating peptide consists of residues 45 through 60 of the 1A protein of RS virus, and it also contains an overlapping antibody binding (B-cell) site. Herein, we have combined the 1A T-cell stimulating peptide with a putative B-cell peptide epitope derived from the viral G glycoprotein using linear synthesis or using chemical crosslinking. The chimeric immunogens were compared to each other and to free peptides for their T- and B-cell stimulating properties. Both chimeras had potent T-cell stimulating and antibody-inducing activity. However, T-cells primed to free peptide differentially recognized the two chimeras and immunization with the chimeras primed T-cells with different specificity. Most strikingly, the two chimeras had opposite antibody-inducing properties: The chimera constructed by linear synthesis overwhelmingly elicited antibody directed against the G peptide, whereas the chimera constructed by chemical crosslinking overwhelmingly elicited antibody directed against the 1A peptide. Competition blocking studies revealed that the chimeras adopted different configurations in solution. The resulting antibody response, and hence the B-cell clone elicited, was consistent with the antibody accessibility of the individual peptide epitope.     

Analysis of T- and B-cell epitopes of capsid protein of rubella virus by using synthetic peptides.

A nested set of 11 overlapping synthetic peptides covering the entire sequence of rubella virus capsid protein was synthesized, purified, and tested against human rubella virus-specific T-cell lines and rubella virus-seropositive sera. T-cell lines derived from four donors responded strongly to four synthetic peptides containing residues 96 to 123, 119 to 152, 205 to 233, and 255 to 280. Only one peptide (residues 255 to 280) was recognized by all four T-cell lines. Two human immunodominant linear B-cell epitopes were mapped to residues 1 to 30 and 96 to 123 by using peptide-specific enzyme-linked immunosorbent assay. All 11 synthetic peptides were highly immunogenic and induced strong antibody responses in rabbits against the respective immunized peptides. Seven of the 11 rabbit antipeptide antisera (anti-1-30, -74-100, -96-123, -119-152, -205-233, -231-257, and -255-280) specifically recognized the capsid protein on immunoblots. Identification of these T- and B-cell epitopes represents the first step toward rational design of synthetic vaccines against rubella.     

SIV envelope glycoprotein epitopes recognized by antibodies from infected or vaccinated rhesus macaques

We analyzed SIV-specific monkey sera to localize B-cell epitopes of the envelope glycoprotein of SIV (gp130), using overlapping synthetic peptides representing the entire SIV gp130 protein and sera from experimentally infected monkeys and monkeys immunized with whole, inactivated SIV. A B-cell epitope which induces neutralizing antibody production and T-cell responses was characterized as well as a new B-cell epitope and a previously described neutralizing epitopes. Vaccinated monkey sera recognize the three epitopes differentially relative to unimmunized controls, and a correlation appears to exist between degree of cross-neutralization by infected monkey sera and degree of binding to these three regions.     

Characterization of a monoclonal antibody reactive with rabbit T lymphocytes and neutrophils

An IgG1 monoclonal antibody, termed ACM-1, has been shown to react with rabbit T cells, but not Ig+ cells or macrophages. This antibody appears to recognize the same epitope as the previously described 9AE10 antibody and, together with 9AE10, has been used to obtain highly pure and fully functional T- and B-cell populations. However, the relevant epitope does not appear to be homologous to rodent Thy-1 since quantitative absorptions failed to show reactivity with rabbit brain. Furthermore, attempts to obtain in vivo T-cell depletion resulted in larger decreases in white blood cells than would be expected for simple T-cell removal. In vitro assays on enriched neutrophil preparations revealed that 80-95% of these cells were reactive with ACM-1 and 9AE10. Thus, it appears that in the rabbit, T cells and neutrophils share a major epitope.     

Interspecies major histocompatibility complex-restricted Th cell epitope on foot-and-mouth disease virus capsid protein VP4

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.     

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C(HCV) genome is highly variable,particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene.The variability of HCV genome has been a major obstacle for de-veloping HCV vaccines.Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes,we synthesized an minigene of HCV-derived multi-epitope peptide an-tigen(CMEP) ,which contains 9 B-cell HVR1 mimotopes in E2,2 conserved CTL epitopes in C,1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3.This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP.The immunogenic properties of CEMP were characterized by HCV infected patients' sera,and found that the reactivity frequency reached 75%.The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%.Meanwhile,we constructed an HCV DNA vaccine candidate,plasmid pVAX1.0-st-CMEP carrying the recombinant gene(st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene.Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody,which was of the same cross reactivity as the fusion protein GST-CMEP.Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes,and would be of the value as a candidate for the development of HCV vaccines.     

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients’ sera, and found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes, and would be of the value as a candidate for the development of HCV vaccines.     

Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.     

Enhancement of the antibody response to flavivirus B-cell epitopes by using homologous or heterologous T-cell epitopes.

We have been investigating the T-helper (Th)-cell response to the flavivirus envelope (E) glycoprotein. In our studies with Murray Valley encephalitis (MVE) virus, we previously identified synthetic peptides capable of priming Th lymphocytes for an in vitro antivirus proliferative response (J. H. Mathews, J. E. Allan, J. T. Roehrig, J. R. Brubaker, and A. R. Hunt, J. Virol. 65:5141-5148, 1991). We have now characterized in vivo Th-cell priming activity of one of these peptides (MVE 17, amino acids 356 to 376) and an analogous peptide derived from the E-glycoprotein sequence of the dengue (DEN) 2, Jamaica strain (DEN 17, amino acids 352 to 368). This DEN peptide also primed the Th-cell compartment in BALB/c mice, as measured by in vitro proliferation and interleukin production. The failure of some MVE and DEN virus synthetic peptides to elicit an antibody response in BALB/c mice could be overcome if a Th-cell epitope-containing peptide was included in the immunization mixture. A more detailed analysis of the structural interactions between Th-cell and B-cell epitope donor peptides revealed that the peptides must be linked to observe the enhanced antibody response. Blockage or deletion of the free cysteine residue on either peptide abrogated the antibody response. The most efficient T-B-cell epitope interaction occurred when the peptides were colinearly synthesized. These Th-cell-stimulating peptides were also functional with the heterologous B-cell epitope-containing peptides. The Th-cell epitope on DEN 17 was more potent than the Th-cell epitope on MVE 17.     

A novel strategy of epitope design in Neisseria gonorrhoeae

In spite of genome sequences of both human and N. gonorrhoeae in hand, vaccine for gonorrhea is yet not available. Due to availability of several host and pathogen genomes and numerous tools for in silico prediction of effective B-cell and T-cell epitopes; recent trend of vaccine designing has been shifted to peptide or epitope based vaccines that are more specific, safe, and easy to produce. In order to design and develop such a peptide vaccine against the pathogen, we adopted a novel computational approache based on sequence, structure, QSAR, and simulation methods along with fold level analysis to predict potential antigenic B-cell epitope derived T-cell epitopes from four vaccine targets of N. gonorrhoeae previously identified by us [Barh and Kumar (2009) In Silico Biology 9, 1-7]. Four epitopes, one from each protein, have been designed in such a way that each epitope is highly likely to bind maximum number of HLA molecules (comprising of both the MHC-I and II) and interacts with most frequent HLA alleles (A*0201, A*0204, B*2705, DRB1*0101, and DRB1*0401) in human population. Therefore our selected epitopes are highly potential to induce both the B-cell and T-cell mediated immune responses. Of course, these selected epitopes require further experimental validation.     

B- and T-lymphocyte responses to an immunodominant epitope of human immunodeficiency virus.

Using synthetic peptides, we characterized the B-lymphocyte (antibody) and T-lymphocyte (proliferation) responses to an immunodominant epitope of human immunodeficiency virus type 1 (HIV-1) located near the amino-terminal end of the transmembrane glycoprotein (env amino acids 598 to 609). Both immunoglobulin M (IgM) and IgG antibodies against this epitope appeared early after primary infection with HIV-1. In an animal model, the IgG response to a synthetic peptide derived from this sequence was T-helper-cell dependent, whereas the IgM response was T-cell independent. In addition, antibody generated by immunization with this peptide had HIV-1-neutralizing activity. Greater than 99% (201 of 203) of patients infected with HIV-1 generated antibody to this peptide in vivo; however, only 24% (7 of 29) had T cells that proliferated in response to this peptide in vitro. These observations suggest that different HIV-1 gp41 epitopes elicit B-cell and T-cell immune responses.     

Identification of B- and T-cell epitopes on HtrA protein of Orientia tsutsugamushi

Orientia tsutsugamushi, a cause of scrub typhus is emerging as an important pathogen in several parts of the tropics. The control of this infection relies on rapid diagnosis, specific treatment, and prevention through vector control. Development of a vaccine for human use would be very important as a public health measure. Antibody and T-cell response have been found to be important in the protection against scrub typhus. This study was undertaken to predict the peptide vaccine that elicits both B- and T-cell immunity. The outer-membrane protein, 47-kDa high-temperature requirement A was used as the target protein for the identification of protective antigen(s). Using BepiPred2 program, the potential B-cell epitope PNSSWGRYGLKMGLR with high conservation among O. tsutsugamushi and the maximum surface exposed residues was identified. Using IEDB, NetMHCpan, and NetCTL programs, T-cell epitopes MLNELTPEL and VTNGIISSK were identified. These peptides were found to have promiscuous class-I major histocompatibility complex (MHC) binding affinity to MHC supertypes and high proteasomal cleavage, transporter associated with antigen processing prediction, and antigenicity scores. In the I-TASSER generated model, the C-score was −0.69 and the estimated TM-score was 0.63 ± 0.14. The location of the epitope in the 3D model was external. Therefore, an antibody to this outer-membrane protein epitope could opsonize the bacterium for clearance by the reticuloendothelial system. The T-cell epitopes would generate T-helper function. The B-cell epitope(s) identified could be evaluated as antigen(s) in immunodiagnostic assays. This cocktail of three peptides would elicit both B- and T-cell immune response with a suitable adjuvant and serve as a vaccine candidate.     

Short synthetic glycopeptides successfully induce antibody responses to carcinoma-associated Tn antigen.

Glycopeptides containing a tumor-associated carbohydrate antigen (mono-, tri- or hexa-Tn antigen) as a B-cell epitope and a CD4+ T-cell epitope (PV: poliovirus or TT: tetanus toxin) were prepared for immunological studies. Several Tn antigen residues [FmocSer/Thr (alpha-GalNAc)-OH] were successively incorporated into the peptide sequence with unprotected carbohydrate groups. The tri- and hexa-Tn glycopeptides were recognized by MLS128, a Tn-specific monoclonal antibody. The position of the tri-Tn motif in the peptide sequence and the peptide backbone itself do not alter its antigenicity. As demonstrated by both ELISA and FACS analysis, the glycopeptides induced high titers of anti-Tn antibodies in mice, in the absence of a carrier molecule. In addition, the generated antibodies recognized the native Tn antigen on cancer cells. The antibody response obtained with a D-(Tn3)-PV glycopeptide containing three alpha-GalNAc-D-serine residues is similar that obtained with the Tn6-PV glycopeptide. These results demonstrate that short synthetic glycopeptides are able to induce anticancer antibody responses.     

Identification and synthesis of the epitope for a human monoclonal antibody which can neutralize human T-cell leukemia/lymphotropic virus type I

A monoclonal antibody (mAb), designated 0.5 alpha, derived from a patient with adult T-cell leukemia was found previously to neutralize the human T-cell leukemia/lymphotropic type I (HTLV-I) virus in in vitro assays and bind to the major envelope glycoprotein (gp46) of HTLV-I (Matsushita, S., Guroff, M.R., Trepel, J., Crossman, J., Mitsuya, H., and Broder, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2671-2676). We have designed experiments to determine the epitope for this mAb. Using simultaneous multiple peptide synthesis, we synthesized 481 overlapping octapeptides which corresponded to the sequence of gp46. We mapped the epitope for mAb 0.5 alpha to lie between residues 186 and 195 of gp46. This result was confirmed by independently synthesizing a peptide containing this epitope which bound specifically to mAb 0.5 alpha with an approximate Ka = 4 x 10(7) M-1. In addition, the peptide inhibited mAb 0.5 alpha binding to gp46 derived from T-cells infected with HTLV-I. This epitope containing peptide may facilitate understanding HTLV-1 infection of T-cells.     

A peptide epitope derived from the cancer testis antigen HOM-MEL-40/SSX2 capable of inducing CD4+ and CD8+ T-cell as well as B-cell responses

Background

Antigen-derived HLA class I-restricted peptides can generate specific CD8⁺ T-cell responses in vivo and are therefore often used as vaccines for patients with cancer. However, only occasional objective clinical responses have been reported suggesting the necessity of CD4⁺ T-cell help and possibly antibodies for the induction of an effective anti-tumor immunity in vivo. The SSX2 gene encodes the cancer testis antigen (CTA) HOM-MEL-40/SSX2, which is frequently expressed in a wide spectrum of cancers. Both humoral and cellular immune responses against SSX2 have been described making SSX2 an attractive candidate for vaccine trials.

Methods

SYFPEITHI algorithm was used to predict five pentadecamer peptides with a high binding probability for six selected HLA-DRB1 subtypes (*0101, *0301, *0401, *0701, *1101, *1501) which are prevalent in the Caucasian population.

Results

Using peripheral blood cells of 13 cancer patients and 5 healthy controls, the HOM-MEL-40/SSX2-derived peptide p101-111 was identified as an epitope with dual immunogenicity for both CD4⁺ helper and cytotoxic CD8⁺ T cells. This epitope also reacted with anti-SSX2 antibodies in the serum of a patient with breast cancer. Most remarkably, SSX2/p101-111 simultaneously induced specific CD8, CD4, and antibody responses in vitro.

Conclusions

p101-111 is the first CTA-derived peptide which induces CD4⁺, CD8⁺, and B-cell responses in vitro. This triple-immunogenic peptide represents an attractive vaccine candidate for the induction of effective anti-tumor immunity.     

Differing T-cell requirements for recombinant retrovirus vaccines.

Friend murine leukemia virus is a retrovirus complex that induces rapid erythroleukemia and immunosuppression in susceptible strains of adult mice. Using this model, we directly examined the T-cell subsets required for a protective retrovirus vaccine. Paradoxically, recovery in mice immunized with a chimeric envelope containing only T-helper (TH) and B-cell epitopes was dependent on CD8+ T cells as well as CD4+ T cells despite the fact that the vaccine contained no CD8+ cytolytic T-lymphocyte (CTL) epitopes. However, the requirement for CD8+ T cells was overcome by inclusion of additional TH and B-cell epitopes in the immunizing protein. These additional epitopes primed for more rapid production of virus-neutralizing antibody which appeared to limit virus spread sufficiently to protect even in the absence of CD8+ T cells. Inclusion of an immunodominant CTL epitope in the vaccine was not sufficient to overcome dependence on CD4+ T cells. These data suggest that TH priming is more critical for retrovirus immunity than CTL priming.     

Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.     

Major histocompatibility complex class II-regulated immunity to murine leukemia virus protects against early T- but not late B-cell lymphomas

We studied the relative importance of class I and class II major histocompatibility complex (MHC) immunoregulation in the control of T- and B-cell lymphomas induced by murine leukemia virus. Previously, we have described a mink cell focus-inducing (MCF) murine leukemia virus, MCF 1233, which induces not only lymphoblastic T-cell lymphomas but also follicle center cell or lymphoblastic B-cell lymphomas. We now report that the outcome of neonatal infection with MCF 1233 in H-2-congenic C57BL/10 and C57BL/6 mice is decisively influenced by the H-2 I-A locus. A total of 64% of H-2 I-Ak, d mice [B10.BR, B10.D2, B10.A(2R), B10.A(4R), and B10.MBR] developed T-cell lymphomas after MCF 1233 infection (mean latency, 37 weeks). In contrast, H-2 I-Ab [B10, B10.A(5R), B6], H-2 I-Ab/k [(B10.A x B10)F1 and (B10 x B10.A)F1], and H-2 I-Abm12 (bm12) mice were resistant against T-cell lymphomagenesis, but 65% of these H-2 I-Ab, b/k, bm12 animals developed B-cell lymphomas (mean latency, 71 weeks). Animals of T-cell lymphoma-susceptible strains that escaped from T-cell lymphomagenesis developed B-cell lymphomas with similar frequency as animals of T-cell lymphoma-resistant strains, but with a shorter latency. H-2 class II-determined regulation of antiviral immunity was reflected in the presence of high titers of antiviral envelope antibodies in T-cell lymphoma-resistant B-cell lymphoma-susceptible H-2 I-Ab, b/k, bm12 mice, whereas in T-cell lymphoma-susceptible H-2 I-Ak,d mice no antiviral antibodies were found. At week 4 after neonatal MCF 1233 infection, a high percentage of thymocytes were virally infected in both T-cell lymphoma-susceptible and -resistant mice. However, T-cell lymphoma-resistant animals cleared the thymic infection between weeks 4 and 10 of age, coinciding with a sharp rise in serum levels of antiviral antibodies. We conclude that the pleiotropic effects of MCF 1233 infection in H-2-congenic mice result from MHC class II I-A-determined T-cell response differences.