Hypervariable epitope constructs as a means of accounting for epitope variability.

Epitope variability is one of the greatest obstacles to development of synthetic peptide vaccines. Based on a recently described hypervariable epitope (aa 414-434) on the envelope glycoprotein (gp130) to simian immunodeficiency virus (SIVmac142), we have developed a novel approach to account for epitope variability. We have prepared, in a single synthesis, a cocktail of peptides, designated a hypervariable epitope construct (HEC), which collectively represent all the in vivo variability seen in an epitope. The HEC represents permutations of amino acid substitutions found in the epitope and has been able to induce antibodies with enhanced binding to native SIV and broad immunoreactivity to related epitope analogues.

An epitope on the surface envelope glycoprotein (gp130) of simian immunodeficiency virus (SIVmac) involved in viral neutralization and T cell activation.

SIVmac infection of macaques is an important animal model for HIV infection and AIDS; this model is being utilized for development of antiviral therapies and vaccines. In the present article, we sought to identify neutralization epitopes of SIVmac envelope surface glycoprotein (gp130). Algorithms were used to predict antigenicity of specific regions. Four regions from the primary amino acid sequence of the viral surface glycoprotein were selected. A synthetic peptide representing one of these regions (414-434) induced virus-neutralizing antibodies in mice; in addition, this peptide induced T cell-proliferative responses in macaques. To address the in vivo relevance of these observations, we demonstrated that experimentally infected macaques produce antibodies to the neutralization epitope. In addition, rhesus macaques protected against infection by an inactivated SIV vaccine develop antibodies that bind to peptide 414-434. These observations demonstrate that the region that includes the sequence 414-434 in the fourth variable domain (V4) of SIVmac gp130 contains both a linear neutralization epitope and a T cell epitope.

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 T cell epitopes on the envelope glycoprotein of simian retrovirus 1 and 2 (SRV-1 and SRV-2) in several mouse strains.

Various mouse strains were immunized with either SRV-1 or SRV-2 virus adsorbed on alum. Seven to 14 days later spleen cells were removed, and spleen cells were cultured with varying amounts of SRV-1 virus and SRV-2 virus, or varying amounts of selected SRV-1 and SRV-2 synthetic envelope peptides to determine their ability to initiate T cell proliferative responses. Our studies demonstrated that all mouse strains tested gave strong proliferative responses with SRV-2 virus. In contrast, SRV-1 virus induced T cell proliferative responses only in H-2k mouse strains. This apparent major histocompatibility complex (MHC)-restriction of SRV-1 virus-induced T cell proliferation correlates with the increased pathogenicity of SRV-1 virus in rhesus monkeys. The SRV envelope peptide 233-249 which is shared by both SRV-1 and SRV-2 virus initiates strong proliferative responses in both SRV-1 and SRV-2 virus immunized mice. The SRV-2 envelope peptide 96-102 initiates significant proliferative responses in SRV-2 immunized mice, and constitutes both a T and B cell epitope. The SRV-2 envelope peptide 127-152 has a 70% homology with the C-terminal region of SRV-1 peptide 142-167. The ability of SRV-2 peptide 127-152 to initiate T cell proliferation in SRV-1 virus immunized mice and the failure of the SRV-1 peptide 142-162 to initiate proliferation suggests that the region encompassing residues 160-167 must represent a T cell epitope in mice immunized with SRV-1 virus.

Immunobiological properties of a recombinant simian retrovirus-1 envelope protein and a neutralizing monoclonal antibody directed against it.

We previously reported that an area encompassing amino acids 147-162 of the envelope region of the simian (type D) retrovirus serotype 1 (SRV-1) constitutes an antigenic site for the binding of murine and rhesus neutralizing antibodies. Neutralizing antibodies to SRV-2 are directed to a different area, encompassing residues 96-102 of SRV-2. This paper presents data on the activity of an SRV-1 recombinant envelope protein (rEP) and of monoclonal hybridoma cell line, C11B8, produced from murine spleen cells immunized with SRV-1 rEP. Purified monoclonal antibodies from C11B8 bind to the SRV-1 rEP and to both SRV-1 and SRV-2. However, the monoclonal antibody exhibits strain specificity in the capacity to neutralize SRV-1 infection in vitro. Thus, C11B8 neutralizes SRV-1 infection but fails to neutralize four other known serotypes of the virus. C11B8 also binds to an SRV-1 synthetic peptide representing residues 142-167, which encompasses the previously defined antigenic site of recognition for neutralizing antibodies to SRV-1. This paper also contains evidence that the SRV-1 rEP construct binds the site for SRV-1 attachment to the cell receptor. This is indicated by the ability of SRV-1 rEP to compete with SRV-1 (but not with SRV-2) and inhibit its infectivity in vitro. In addition, SRV-1 rEP inhibits the neutralizing activity of C11B8 against SRV-1 infection in vitro. SRV-1 rEP has no inhibitory effect on rhesus neutralizing antibodies to SRV-2. Taken together, the above findings indicate that immunity conferred at the level of neutralizing antibodies during SRV infection is strain-specific and involves the recognition of envelope sequences unique to each strain.

The induction of neutralizing antibodies by synthetic peptides of the envelope protein of type D simian retrovirus-1 (SRV-1).

It has been recently demonstrated that two serotypes of type D simian retroviruses, namely SRV-1 and SRV-2, exhibit extensive immunological cross-reactivity but do not exhibit cross-reactivity at the level of neutralizing antibodies. We have also shown recently that an area which includes residues 147-162 of the envelope protein of SRV-1 constitutes an epitope to which neutralizing antibodies against SRV-1 but not against SRV-2 are directed. However, in spite of the capacity of various immunogenic preparations to induce antibodies which react with SRV-1 these antibodies were incapable of neutralizing in vitro viral infectivity. Work reported herein demonstrates that various immunogens consisting of a larger peptide, namely 142-167 of the envelope protein of SRV-1, induce antibodies capable of binding with the envelope protein of SRV-1 and with the whole virus. Moreover, these antibodies exhibit the capacity to neutralize in vitro the infectivity of SRV-1 but not of SRV-2.

Characterization of T- and B-cell epitopes of a simian retrovirus (SRV-2) envelope protein.

Synthetic envelope peptides of a simian retrovirus (SRV-2) were used to define both T- and B-cell epitopes of the envelope protein. The SRV-2 peptide 100-106 specifically blocks rhesus anti-SRV-2 neutralizing antibody activity, and a peptide 100-106 keyhole limpet hemocyanin conjugate induces a strong antipeptide antibody response. SRV-2 peptide 100-106 and 233-249 induces good T-cell proliferation of murine spleen cells immunized with the SRV-2 virus. Thus, SRV-2 envelope peptide 100-106 represents both a T- and B-cell epitope, and peptide 233-249 a T-cell epitope.

Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness.

We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells.

Isolation and characterization of the neutralizable epitope of simian retrovirus-1 (SRV-1) and of the cell receptor for the virus.

An area encompassing residues 142-167 of the envelope protein of type D simian retrovirus (SRV-1) has been shown to contain the epitope to which neutralizing antibodies are directed. This area has been synthesized and shown to bind to monkey and mouse antiviral antibodies and to a virus neutralizing mouse monoclonal antibody. Protein conjugates of this peptide as well as the cross-linked or the free peptide induce antibodies capable of neutralizing, in vitro, viral infectivity. The cell receptor to the virus was isolated following extraction of Raji cells with non-ionic detergents. The receptor was isolated and characterized following radioimmuno-precipitation of 125I labeled cell extract bound to viral envelope protein. This immunoprecipitation could be inhibited by antiserum to peptide 142-167. Analysis in gels indicate that the receptor is of molecular weight of approximately 60 KDa. These results indicate that the neutralizing antibodies and the receptor recognize the same area on the viral envelope protein and that neutralization is the result of blocking the virus-receptor interaction by antibodies.

Synthetic peptides of envelope proteins of two different strains of simian AIDS retrovirus (SRV-1 and SRV-2) represent unique antigenic determinants for serum neutralizing antibodies.

There are at least three distinct serotypes of simian type D retrovirus (SRV) which exhibit extensive serological cross-reactivity, but no cross-reactivity exists at the level of serum neutralizing antibodies. Amino acid sequence analysis and hydrophobicity plots of SRV-1 and SRV-2 envelope proteins were compared in order to identify unique potential antigenic determinants to which respective neutralizing antibodies may be directed. Peptides representing residues 147-162 of SRV-1 and 96-102 of SRV-2 were synthesized and assessed for their immunoreactivity. Free peptide inhibition of strain-specific serum (rhesus) neutralizing antibodies to SRV-1 and SRV-2 was demonstrated using the SRV-1 147-162 peptide and the SRV-2 peptide, 96-102, respectively. Inhibition of serum neutralizing activity by these peptides was also strain-specific, showing no cross-inhibition. SRV-1 147-162 conjugated to a protein carrier and cross-linked to Sepharose beads specifically adsorbed neutralizing antibodies from SRV-1 immune rhesus sera. The antibodies eluted from the immunoadsorbent possessed SRV-1 neutralizing activity, but showed no effect on the infectivity of SRV-2. Peptide SRV-1 147-162 also exhibited the capacity to bind specifically with a mouse monoclonal antibody which neutralizes the infectivity of SRV-1. Mice immunized with a recombinant SRV-1 envelope protein or with whole, inactivated SRV-1 produced antibodies which bound the SRV-1 147-162 conjugate, but not the protein carrier itself. Mouse antibodies to the SRV-1 147-162 conjugate exhibited specific binding with both native SRV-1 and with recombinant SRV-1 envelope protein. These findings provide strong evidence that SRV-1 147-162 and SRV-2 96-102 constitute at least two unique antigenic determinants, or parts thereof, which participate in the strain-specific neutralizing antibody response. Moreover, the findings indicate that the SRV-1 neutralizing antibodies produced by monkeys and at least a certain population of neutralizing antibodies produced by mice recognize the same epitope of SRV-1.

Diverse VH and VL genes are used to produce antibodies against a defined protein epitope.

mAb secreting hybridomas were produced from mice hyperimmune to the model Ag tobacco mosaic virus protein. Six mAb were selected for their ability to bind synthetic peptides corresponding to amino acid residues 103-112 and 97-107 of tobacco mosaic virus protein. These mAb were analyzed for their fine specificity by measuring binding to synthetic analogs of the decapeptide, and cDNA sequences encoding the mAb V regions were determined. These analyses revealed that a wide range of different V regions are capable of binding with the same decapeptide epitope, and that these antibody sequence differences generally coincided with different binding fine specificities. This diverse antibody response with specificity for the same epitope demonstrates both the breadth of potential of the immune system and the lack of exclusivity in specific protein:protein interactions.

In vivo-induced suppression of T cell proliferation: the relationship between the specificity of induction and control.

We have previously shown that sc immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and to peptide 8. In contrast, immunization of B10.BR (H-2k) mice either with TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous Ag. In the present article , we report that in the B10.BR (H-2k) strain, ip prepriming with (TMVP) 7 days prior to sc immunization with peptide 8 causes a drastic reduction in the in vitro proliferative response of peptide 8-specific T cells while no such effect is seen in the congenic C57BL/10 (H-2b) strain. This suppression of T cell responsiveness can be transferred with TMVP-primed spleen cells. Moreover, deleting T cells from the transferred spleen cells abrogates the suppressive effect. In both H-2 haplotypes, ip prepriming with peptide 8 causes suppression of the proliferative T cell response induced by subsequent immunization with peptide 8. This prepriming has no effect on the response to TMVP immunization of B10.BR mice but does effect the response of C57BL/10 mice. Using various synthetic peptides to analyze the specificity of the suppression, we have determined that (1) T cells involved in the suppression of the proliferative T cell response to a single peptide determinant do not suppress the proliferative T cell response to other determinants on the protein antigen and (2) these T cells with suppressor function, and proliferating T cells which are ultimately regulated, can exhibit specificity for the same epitope. These studies suggest that there may exist fundamental differences as to how T cells which participate in suppression an proliferating T cells (which include mainly T helper cells) recognize protein antigens.

Bluetongue virus-17 fusion protein ns1 expressed in Escherichia coli by pUC vectors.

The cDNA coding for the major nonstructural protein, NS1, of bluetongue serotype 17 (BTV-17) was cloned previously. Using pUC plasmids, we have successfully expressed the NS1 protein in Escherichia coli as a LacZ-NS1 fusion protein. The recombinant NS1 protein reacted with rabbit anti-BTV-17 antiserum, and was thus immunologically indistinguishable from the native BTV-17 NS1 protein. This was the first bluetongue viral protein to be produced in a bacterial system.

Structural features of an antigen required for cellular interactions and for T cell activation in a MHC-restricted response.

The protein Ag, tobacco mosaic virus protein, (TMVP) and its tryptic peptide number 8 (residues 93-112 of the protein) exhibit cross-reactivity on the T cell level in some strains of mice (e.g., C3H.SW, C57BL/10); these strains are termed cross-reactive (CR). In other strains such as A/J or B10.BR, no cross-reactivity is exhibited; these strains are termed non-cross-reactive (NCR). Genetic experiments indicated that the cross-reactivity is dominant and that it is mapped to the I-A or I-E region of the MHC, with cross-reactivity exhibited by the I-Ab haplotype but not by I-Ak or I-Ek. Cell reconstitution experiments have indicated that the non-cross-reactivity is associated with the inability of the NCR APC to present Ag. Analysis of the area(s) on peptide 8 which serve(s) as epitope revealed that both strains recognize an overlapping area consisting of 11 amino acid residues in the middle of peptide 8 (residues 97-107), which by itself is nonstimulatory to TMVP- or peptide 8-immune T cells of the CR or the NCR strains. However, the addition of a few amino acid residues of the sequence of peptide 8 to this area converts it to a complete stimulatory epitope. Additivity experiments revealed that the CR strain contains two major T cell populations each recognizing this middle region of peptide 8 when elongated by a few amino acids N-terminally and C-terminally, respectively. In contrast, the NCR strain contains one major T cell population recognizing elongation only N-terminally. Because TMVP (but not peptide 8) requires processing before presentation to T cells, it is postulated that, during processing of TMVP, there occur alterations in the area of the proximal three or four N-terminal amino acids of the region consisting of peptide 8, destroying the only region containing the T cell epitope recognized by the NCR strain, hence TMVP and peptide 8 do not exhibit cross-reactivity in this strain. The same alterations of TMVP still leave intact an epitope consisting of amino acid residues C-terminal to the altered area which is recognized by the CR strain, hence the cross-reactivity exhibited by this strain. The results suggest that the difference in cross-reactivity on the T cell level between TMVP and peptide 8 exhibited by the strains may be due to differences in the orientation of presentation and the subsequent cell recognition of an epitope contained within peptide 8.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on the clonality of the response to an epitope of a protein antigen. Randomness of activation of epitope-recognizing clones and the development of clonal dominance.

Syngeneic mice immunized with tobacco mosaic virus protein (TMVP) can differ with respect to their ability to produce antibodies that bind a decapeptide epitope representing residues 103 to 112 of TMVP, and with respect to the fine specificity of the decapeptide binding antibodies as determined by their ability to bind several synthetic analogues of the decapeptide. To elucidate the mechanism responsible for the differences between the syngeneic animals in their ability to make anti-decapeptide antibodies, spleen cells from a large number of naive CSW mice were pooled, and aliquots were transferred (either including or excluding resident T cells) into naive recipients that were subsequently immunized with TMVP. Examination of the frequency and fine specificity of anti-decapeptide antibodies revealed that the recipients exhibited various clonalities of decapeptide binding antibody responses similar to those seen in a normal population of CSW mice. Moreover, the response of each individual mouse was of a restricted clonality despite the availability of a more extensive repertoire of decapeptide-recognizing clones. The results indicate that the selection of the clonality of the antibody response was not determined by the presence (or absence) of particular clones of B or T cells and that the mechanism responsible for generating differences between mice must have acted, subsequent to introduction of the Ag, by activation of a limited number of clones randomly selected by Ag and/or by Ag-driven mutation. The long term nature of the antibody response to the decapeptide epitope was also investigated. The response was shown to be "locked-in" for the life of the immunized individual. Thus, individuals that responded to TMVP but that did not produce antibodies to the decapeptide after the first set of immunizations with TMVP maintained their non-responsiveness to the decapeptide after the second set of immunizations with the protein. However, individuals that responded to an initial set of immunizations with TMVP by producing antibodies to the decapeptide epitope continue to produce antibodies to the decapeptide after a second set of immunizations with TMVP. The fine specificity of the decapeptide-binding antibodies also appeared to be "locked in" throughout the life of the immunized individual. The long term maintenance of the clonability of the antibody response does not appear to be influenced by Ag-specific T cells and is strictly a function of memory B cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on the suppression of the immune response to a defined protein epitope by anti-idiotypic antibodies.

We have previously reported that C3H.SW (CSW) and A/J mice immunized with the tobacco mosaic virus protein (TMVP) produce antibodies to a decapeptide epitope corresponding to amino acid residues 103-112 of the protein. In the C3H.SW (CSW) strain, the antibodies to the decapeptide contain major crossreactive idiotope, C10-IdX, which is found on a CSW-derived monoclonal antidecapeptide antibody, designated as C10. The in vivo administration of anti-C10 antibodies suppresses the response to the decapeptide epitope in CSW mice. The present communication describes experiments designed to elucidate several parameters responsible for the suppression produced by the in vivo administration of anti-C10. It was found that 50 micrograms of anti-C10 was required to suppress the response to the decapeptide in CSW mice when 2 weeks elapsed between administration of the anti-C10 and immunization with TMVP; however, only 10 ng was required when one injection of antigen instead of two was administered and when the interval between treatment with anti-C10 and immunization was extended to 6 weeks. This suggests that the anti-C10 induces alterations in the idiotypic network which are not yet fully developed after 2 weeks. Furthermore, experiments presented herein demonstrate that decapeptide-binding antibodies from A/J mice, which lack the C10-IdX, were also suppressed by pretreatment with anti-C10. Interestingly, unlike the case with the CSW strain, the titer to TMVP was decreased by the administration of anti-C10 to A/J mice which were subsequently immunized with TMVP. These findings suggest that the polyclonal anti-C10 contains antibodies to an idiotype which is a major component of the overall anti-TMVP response of A/J mice and may be important in the overall regulation of the anti-TMVP response.

Differential reactivity of rheumatoid synovial cells and serum rheumatoid factors to human immunoglobulin G subclasses 1 and 3 and their CH3 domains in rheumatoid arthritis.

19S IgM rheumatoid factors (RF) are polyclonal autoantibodies that may play an important pathogenic role in sustaining inflammatory synovitis in rheumatoid arthritis (RA). RF in RA have reactivity for as-yet-uncharacterized antigenic determinants in IgG Fc. We hypothesized that qualitative differences might exist between some of these RF molecules, and that differences such as reactivity and affinity might characterize more pathogenic RF molecules. Previous observations in our laboratory indicate that RF produced by rheumatoid synovial cells (RSC) have greater reactivity with human IgG and IgG3 subclass, in contrast to serum RF, which has greater reactivity with rabbit IgG and human IgG1. These observations were made using a complement-dependent RF plaque-forming cell assay. The purpose of this study was to validate and extend those observations. Therefore, we examined the reactivity of RSC and serum RF with human and rabbit IgG and the reactivity and avidity of RSC-RF for IgG1 and IgG3 molecules and Fab, F(ab')2, and pFc' fragments thereof in a solid-phase enzyme immunoassay. In particular, we found: RSC-RF had at least twice as much reactivity with human IgG as with rabbit IgG; serum RF had approximately equal reactivity with human and rabbit IgG; RSC-RF had greater reactivity and avidity for IgG3 and IgG3 pFc' than for IgG1; and RSC-RF was nonreactive with Fab or F(ab')2 from either IgG1 or IgG3. These results suggest that the major antigenic determinant for RSC-RF resides in the CH3 domain of the IgG3 molecule. Precise characterization of this epitope may provide further insight into the etiology and pathogenesis of RA.

Resistance of California ground squirrels (Spermophilus beecheyi) to the venom of the northern Pacific rattlesnake (Crotalus viridis oreganus): a study of adaptive variation.

Recent studies have documented natural resistance to snake venom in a number of diverse mammalian species. The present paper documents for the first time variation in such resistance within one single species, the California ground squirrel (Spermophilus beecheyi). This species is a frequent prey of the northern Pacific rattlesnake (Crotalus viridis oreganus) in certain habitats. Venom resistance was tested directly in two populations of ground squirrels by injection of 1-40 mg/kg venom doses. One population was obtained from a habitat with a high rattlesnake density; the other population came from a rattlesnake-free habitat. Dramatic differences in the response to venom between these populations were manifested, based on a variety of criteria, such as mortality, necrosis and healing time. Resistance to venom was also examined by LD50 tests in groups of mice pre-injected with ground squirrel sera from three rattlesnake-adapted California populations and a non-adapted Arctic population (S. parryii) from snake-free central Alaska. The California ground squirrel sera were 3.3-5.3 times more effective in the in vivo neutralization of venom than the sera from Arctic ground squirrels. Moreover, the level of protection by the sera as reflected by the LD50 values was highly correlated (P less than 0.005) with the level of in vitro squirrel serum-venom binding as quantified by radioimmunoassay (RIA). A subsequent RIA revealed that binding levels of sera from 14 California ground squirrel populations correlated significantly (P less than 0.025) with local rattlesnakes densities; i.e. sera pools from populations sympatric with rattlesnakes exhibited the highest binding, whereas populations living in habitats where rattlesnakes are rare or absent typically exhibited the lowest binding levels, several of which approximated the Arctic control. Taken together, these results demonstrate intraspecific variation that is probably the result of differential natural selection due to northern Pacific rattlesnakes. This intraspecific variation should be taken into consideration when testing for natural resistance in wild-caught species.