Immunogenic Ones Research Articles

A hidden element governing immunogenicity in tumoral neoepitopes

The search for what characteristics define an epitope as either an immunogenic or a non-responsive target for immunotherapy has eluded researchers for years. Several studies demonstrate that certain positions in the peptide sequences, the major histocompatibility complex (MHC) anchor residues, have a preferential composition of amino acids (allelic motifs), being those epitopes more likely to display a better immunogenic response. First of all, not all MHC ligands are immunogenic, considering that unnumbered self-epitopes are continuously presented on the cell surfaces. In this work, we analyzed data to evaluate an additional element, central to our hypothesis that alterations in tumor protein sequences result in a structural change that shifts the electrostatic surface of the peptide-major histocompatibility complex (pMHC) molecules, pivotal for T-Cell receptor (TCR) recognition and the initiation of an immunogenic response. Firstly, previously neoepitope sequences presenting differential immune responses when compared with their wild-type counterparts were recovered. Even though the sequences were very similar, they triggered responses that were considerably different, and currently, there is no well-established explanation for why they conspicuously differ in immunogenic aspects from each other. The pMHCs structures harboring the epitope sequences were modeled and then used to generate images of their electrostatic surfaces, looking for qualitative differences that indicate the distinct responses. We noticed that no significant alteration occurred between immunogenic tumor peptides and their wild-type non-immunogenic counterparts when comparing their electrostatic surface. An additional comparison was made against structures of pMHCs containing immunogenic epitopes recovered from the Crosstope Database (https://crosstope.com.br/). In this sense, it was also possible to verify if immunogenic tumor epitopes were similar to viral immunogenic ones. Surprisingly, both wild-type (WT) sequences and neoepitopes shared an electrostatic surface distribution with pathogen targets, which could indicate their immunogenic predisposition. So we theorized that a “hidden element” may be responsible for the immunogenicity shift in neoepitopes.

Abstract 1707: Increasing immune cell infiltration in hepatocellular carcinoma tumors using a novel GPC3-targeting aptamer conjugate

Abstract Glypican 3 (GPC3) is a cell membrane-bound heparin sulfate proteoglycan, expressed at the cell surface of many cancer types. It has emerged as a leading target for hepatocellular carcinoma treatment due to its overexpression in aberrant liver cells and low expression in healthy adult tissues. One of the major challenges in treating hepatocellular carcinoma with current immunotherapies is turning cold tumors into immunogenic ones. Unmethylated CpG oligodeoxynucleotides (ODNs) are known immune stimulators, however their use in combination therapy for cancer treatment is limited due to the requirement of intra tumoral injections to prevent systemic toxicity. Here, we describe a unique GPC3-specific aptamer, able to target GPC3-expressing cancer cells in vivo as a means to deliver the immunostimulatory CpG 7909. Systemic administration through intra peritoneal (i.p.) injection of the GPC3 aptamer-CpG conjugate (GRX51) decreases Hep3B tumor volume in a Balb/c nude mouse xenograft model by ~30-50% depending on treatment schedule, while also increasing immune cell infiltration. Final tumor volumes of GRX51-treated animals are consistent with tumor volumes of animals treated with the standard of care compound, sorafenib tosylate. Due to its GPC3-targeting capacity, systemic dosing of GRX51 (15 mg/kg) is less toxic than systemic administration with equimolar amounts of CpG 7909 alone (5 mg/kg), with a comparable effect on tumor shrinkage. While both GRX51 and CpG 7909 induce immune cell recruitment, GRX51 treated mice had a final immune:tumor cell ratio of 2.45 ± 0.56, while CpG 7909-treated animals had a ratio of 1.51 ± 0.95 relative to the vehicle control after a 7-day on/7-day off daily dosing schedule. Using the Hep3B xenograft in CD34+ humanized mice, we also showed GRX51 primes tumors for combination treatments with FDA approved checkpoint inhibitors. Mice were treated with GRX51 (7.5 mg/kg daily i.p. dose, 11 days), the PD-1R inhibitor pembrolizumab (10 mg/kg once every 3 days i.p., 11 days), or a combination of the two drugs. The GRX51 treatment alone outperformed pembrolizumab alone, reducing tumor size by 33% and 18% respectively. Notably, the combination of GRX51 with pembrolizumab reduced tumor size by 63%. Together, our data show that GRX51 is a novel GPC3-targeting molecule capable of reducing tumor size and priming GPC3-positive tumors for combination immunotherapy, without inducing major systemic toxicity with i.p. injection. Citation Format: Christina McGuire, Jin Yuan, Aaron Ball, Justin Sonberg, Yuxun Wang, Kristin Thompson, Shuhao Zhu. Increasing immune cell infiltration in hepatocellular carcinoma tumors using a novel GPC3-targeting aptamer conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1707.

TruNeo: an integrated pipeline improves personalized true tumor neoantigen identification

BackgroundNeoantigen-based personal vaccines and adoptive T cell immunotherapy have shown high efficacy as a cancer treatment in clinical trials. Algorithms for the accurate prediction of neoantigens have played a pivotal role in such studies. Some existing bioinformatics methods, such as MHCflurry and NetMHCpan, identify neoantigens mainly through the prediction of peptide-MHC binding affinity. However, the predictive accuracy of immunogenicity of these methods has been shown to be low. Thus, a ranking algorithm to select highly immunogenic neoantigens of patients is needed urgently in research and clinical practice.ResultsWe develop TruNeo, an integrated computational pipeline to identify and select highly immunogenic neoantigens based on multiple biological processes. The performance of TruNeo and other algorithms were compared based on data from published literature as well as raw data from a lung cancer patient. Recall rate of immunogenic ones among the top 10-ranked neoantigens were compared based on the published combined data set. Recall rate of TruNeo was 52.63%, which was 2.5 times higher than that predicted by MHCflurry (21.05%), and 2 times higher than NetMHCpan 4 (26.32%). Furthermore, the positive rate of top 10-ranked neoantigens for the lung cancer patient were compared, showing a 50% positive rate identified by TruNeo, which was 2.5 times higher than that predicted by MHCflurry (20%).ConclusionsTruNeo, which considers multiple biological processes rather than peptide-MHC binding affinity prediction only, provides prioritization of candidate neoantigens with high immunogenicity for neoantigen-targeting personalized immunotherapies.

Microbial Transglutaminase Is Immunogenic and Potentially Pathogenic in Pediatric Celiac Disease.

The enzyme microbial transglutaminase is heavily used in the food processing industries to ameliorate food qualities and elongate the products' shelf life. As a protein's glue, it cross-links gliadin peptides, creating neo-complexes that are immunogenic and potentially pathogenic to celiac disease communities. Even lacking sequence identity, it imitates functionally the endogenous tissue transglutaminase, known to be the autoantigen of celiac disease and representing an undisputable key player in celiac disease initiation and progress. The present review expend on the enzyme characteristics, exogenous intestinal sources, its cross-linking avidity to gluten or gliadin, turning naïve protein to immunogenic ones. Several observation on microbial transglutaminase cross linked complexes immunogenicity in celiac patients are reviewed and its pathogenicity is summarized. Warnings on its potential risks for the gluten dependent conditions are highlighted. When substantiated, it might represent a new environmental factor of celiac disease genesis. It is hoped that the presented knowledge will encourage further research to explore the mechanism and the pathogenic pathways taken by the gliadin cross linked enzyme in driving celiac disease.

Neoantigens Generated by Individual Mutations and Their Role in Cancer Immunity and Immunotherapy.

Recent preclinical and clinical studies have proved the long-standing hypothesis that tumors elicit adaptive immune responses and that the antigens driving effective T-cell response are neoantigens, i.e., peptides that are generated from somatically mutated genes. Hence, the characterization of neoantigens and the identification of the immunogenic ones are of utmost importance for improving cancer immunotherapy and broadening its efficacy to a larger fraction of patients. In this review, we first introduce the methods used for the quantification of neoantigens using next-generation sequencing data and then summarize results obtained using these tools to characterize the neoantigen landscape in solid cancers. We then discuss the importance of neoantigens for cancer immunotherapy using checkpoint blockers, vaccination, and adoptive T-cell transfer. Finally, we give an overview over emerging aspects in cancer immunity, including tumor heterogeneity and immunoediting, and give an outlook on future prospects.

Combination of glycolysis inhibition with chemotherapy results in an antitumor immune response

Most DNA-damaging agents are weak inducers of an anticancer immune response. Increased glycolysis is one of the best-described hallmarks of tumor cells; therefore, we investigated the impact of glycolysis inhibition, using 2-deoxyglucose (2DG), in combination with cytotoxic agents on the induction of immunogenic cell death. We demonstrated that 2DG synergized with etoposide-induced cytotoxicity and significantly increased the life span of immunocompetent mice but not immunodeficient mice. We then established that only cotreated cells induced an efficient tumor-specific T-cell activation ex vivo and that tumor antigen-specific T cells could only be isolated from cotreated animals. In addition, only when mice were immunized with cotreated dead tumor cells could they be protected (vaccinated) from a subsequent challenge using the same tumor in viable form. Finally, we demonstrated that this effect was at least partially mediated through ERp57/calreticulin exposure on the plasma membrane. These data identify that the targeting of glycolysis can convert conventional tolerogenic cancer cell death stimuli into immunogenic ones, thus creating new strategies for immunogenic chemotherapy.

Direct activation of antigen-presenting cells is required for CD8+T-cell priming and tumor vaccination

Successful priming of adaptive immune responses is crucially dependent on innate activation signals that convert resting antigen-presenting cells (APCs) into immunogenic ones. APCs expressing the relevant innate pattern recognition receptors can be directly activated by pathogen-associated molecular patterns (PAMPs) to become competent to prime T-cell responses. Alternatively, it has been suggested that APCs could be activated indirectly by proinflammatory mediators synthesized by PAMP-exposed cells. However, data obtained with CD4(+) T cells suggest that inflammatory signals often cannot substitute for direct pattern recognition in APC activation for the priming of T helper responses. To test whether the same is true for CD8(+) T cells, we studied cytotoxic T lymphocyte development in vitro and in mixed chimeric mice in which coexisting APCs can either present a preprocessed model antigen or directly recognize a given PAMP, but not both. We show that indirectly activated APCs promote antigen-specific proliferation of naïve CD8(+) T cells but fail to support their survival and cytotoxic T lymphocyte differentiation. Furthermore, CD8(+) T cells primed by indirectly activated APCs are unable to reject tumors. Thus, inflammation cannot substitute for direct recognition of single PAMPs in CD8(+) T-cell priming. These findings have important practical implications for vaccine design, indicating that adjuvants must be judiciously chosen to trigger the relevant pattern recognition receptors in APCs.

Optimal TLR9 signal converts tolerogenic CD4–8– DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses

Abstract TLR ligands have been reported to promote DC maturation and enhance CD8+ CTL responses. We have demonstrated previously that CD4-8- DCs secreting TGF-beta stimulate CD4+ Tr1 cell responses. Here, we have assessed whether TLR4 and TLR9 signaling through LPS and CpG stimulation can convert CD4-8- DC-induced tolerance. We demonstrate that immature OVA-pulsed CD4-8- DCs cultured in medium with LPS (2 microg/ml) and CpG (5 microg/ml) for 8 h became mature DCs (DCOVA) with no TGF-beta secretion. CpG-treated, CD4-8- DCOVA-secreting IL-6/IL-15 induced IFN-gamma/IL-17-secreting/T-bet- and ROR-gammat-expressing CD4+ Th1/Th17, whereas LPS-treated CD4-8- DCOVA stimulated IFN-gamma-secreting/T-bet-expressing CD4+ Th1 responses. The former also significantly stimulated more efficient OVA-specific CD8+ T cell responses and antitumor immunity against OVA-expressing BL6-10OVA tumor cells than the latter (P<0.05). CpG-treated, CD4-8- DCOVA-stimulated CD4+ Th1/Th17 cell responses and antitumor immunity were found to be reduced by using neutralizing anti-IL-6, IL-15, and NK1.1 antibodies in wild-type C57BL/6 mice, IL-15R-/- mice for immunization, or CD4-8- (IL-6-/-) DCOVA for immunization in C57BL/6 mice. Interestingly, in vitro-generated CD4+ Th17 cells significantly enhanced LPS-treated, CD4-8- DCOVA-induced in vivo antitumor immunity via increasing CD8+ CTL responses (P<0.05), although they did not show any direct killing activity against tumor cells in vitro. In addition, prolonged 48 h CpG-treated CD4-8- DCOVA dramatically diminished its cytokine secretion, stimulatory effect, and antitumor immunity. Taken together, our data demonstrate an effect of conversion of tolerogenic DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses by optimal CpG signaling, which may advance current understanding of the importance of TLR9 signaling in a DC-based cancer vaccine.

Pattern of recognition of Besnoitia besnoiti tachyzoite and bradyzoite antigens by naturally infected cattle

Bovine besnoitiosis is caused by the protozoan parasite Besnoitia besnoiti. Many recent cases have been described in different European countries, which may be indicative of expansion of the disease in the next few years. Many infected animals remain asymptomatic; therefore, serological tests are essential tools for diagnosis. The objective of the present work was to identify B. besnoiti tachyzoite and bradyzoite immunodominant antigens (IDAs). IDAs were recognised by SDS-PAGE under reducing conditions and Western blot analysis. Positive sera from symptomatic ( n = 18) and asymptomatic ( n = 18) cattle came from herds endemically infected by B. besnoiti and were confirmed positive by IFAT, whereas negative sera ( n = 4) came from besnoitiosis-free herds and were also confirmed negative by IFAT. Up to 28 tachyzoite antigens in the range of 8.5–190.8 kDa were recognised. Based on the frequency of recognition, six IDAs (14.2, 33, 37.1, 39.6, 46.3 and 190.8 kDa) were identified. The 37.1 kDa antigen was recognised by 100% of sera, usually as an intense band. On the other hand, 30 bradyzoite antigens in the range of 8.5–187.9 kDa were detected. Seven bradyzoite IDAs (8.5, 15.1, 16.8, 19.0, 34.7, 38.6 and 124.4 kDa) were identified and two of them (15.1 and 16.8 kDa) were considered the most immunogenic ones. Additionally, sera from animals with clinical symptoms recognised a significantly higher number of bradyzoite antigens. Finally, significant cross-reactions with other closely related apicomplexan parasites were not detected. This is the first description of B. besnoiti bradyzoite antigens. In addition, the identification of tachyzoite and bradyzoite IDAs may be useful for the development of vaccines and diagnostic tools for differentiating between acute and chronic infections. Further proteomic studies are needed in order to identify stage-specific proteins.

CD40 ligation converts TGF-β-secreting tolerogenic CD4 −8 − dendritic cells into IL-12-secreting immunogenic ones

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in maturation and activation of DCs leading to induction of immune responses. Our previous studies showed that the mouse splenic CD4 −8 − DCs are tolerogenic and capable of stimulating suppressive type 1 CD4 + regulatory T (Tr1) cell responses via TGF-β secretion. In this study, we investigated whether CD40 ligation is able to convert tolerogenic CD4 −8 − DCs into immunogenic ones by in vitro treatment of DCs with anti-CD40 antibody. Our data showed that in vitro CD40 ligation with anti-CD40 antibody converted TGF-β-secreting tolerogenic CD4 −8 − DCs into IL-12-secreting immunogenic ones capable of stimulating type 1 CD4 + helper T (Th1) and CD8 + cytotoxic T lymphocyte (CTL) responses leading to induction of antitumor immunity. In addition, in vivo CD40 ligation by intratumoral injection of adenoviral vector AdVCD40L expressing CD40 ligand also induced tumor growth inhibition and regression of established P815 tumors with infiltration of tolerogenic CD4 −8 − DCs. Therefore, our data provide new information for and may thus have useful impacts in CD40 ligation-based immunotherapy of cancer.

TLR ligands efficiently convert tolerogenic CD4‐8‐ DC into immunogenic ones stimulating long‐lasting antitumor immune response

Different dendritic cell (DC) subsets play distinct roles in immune responses. CD4−8−DC secreting transforming growth factor (TGF)‐β stimulate CD4+ regulatory T type 1 (Tr1) cell responses leading to antitumor immune tolerance. CD4−8−DC could be converted into inducing Th1 rather Tr1 by being cultured for another 16–24 hrs and pulsed with 1 mg/ml OVA protein. In this study we investigate whether TLR ligands, agonist anti‐CD40 and their combinations could more efficiently convert CD4−8−DC to induce Th1 response. We found that CpG, LPS and anti‐CD40 alone or CpG+anti‐CD40, but not LPS+anti‐CD40 stimulate those DCs to express similar higher level of CD40, CD54, CD80, CD86 and I‐Ab after 8hrs culture than newly isolated DCs; Among the above groups, CpG group showed the strongest capacity of stimulating the proliferation of OT1 CD8 and OTII CD4 T cells and induced 100% animal protection from tumor challenge with highest OVAI specific CTL ratio and in vivo cytotoxicity; LPS+anti‐CD40 group was the weakest in every above aspect and could not provide any animal protection. Taken together, our findings indicate that CpG 8 hrs treatment could convert tolerogenic DC into immunogenic ones provoking strong antitumor immunity; and TLR ligands plus anti‐CD40 combination should be carefully tested before using as adjuvant for DC maturation. Thus our results might have great impact on designing the future tumor vaccine.

Conversion of Tolerogenic CD4¯8¯ Dendritic Cells to Immunogenic Ones Inducing Efficient Antitumor Immunity

Culturing conditions may affect dendritic cell (DC) maturation status and functional effects. We have previously demonstrated that different DC subsets play distinct roles in immune responses. The splenic CD4-8- DC subset that secretes transforming growth factor (TGF)-beta stimulates CD4+ regulatory T type 1 (Tr1) cell responses, and this leads to antitumor immune tolerance. In this study, we investigated the potential effect of culturing conditions, namely: (1) duration of culturing and (2) the dose of antigen ovalbumin (OVA) for DC pulsing, respectively, in the conversion of tolerogenic CD4-8- DC into immunogenic DCs. Our data showed that isolated CD4-8- DCs cultured for an additional 18 hours in medium containing 15-20 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) became more mature compared to the freshly isolated CD4-8- DCs. When pulsed with OVA at the relatively high concentration of 1 mg/mL, but not at 0.1 mg/mL, the CD4-8- DCs could be converted into immunogenic CD4-8- DCs, which stimulated CD4+ T-cell differentiation into type 1 helper T (Th1) cells. Vaccination of mice with converted CD4-8- DCs induced strong OVA-specific cytotoxic T-lymphocyte (CTL) responses and protective immunity against OVA-expressing BL6-10OVA B16 melanoma. Taken together, our findings indicate that the conversion of DCs from a tolerogenic to an immunogenic state can be achieved by the elongation of DC culturing time in combination with a high-dose antigen for DC pulsing. Therefore, our results may have a significant impact in designing DC-based antitumor vaccines.

The identification of immunodominant linear epitopes of dengue type 2 virus capsid and NS4a proteins using pin-bound peptides

We have used multi-pin peptide synthesis strategy to identify B-cell epitopes on two small dengue virus proteins, capsid and NS4a. We have identified several linear, immunodominant epitopes on both these proteins. Almost all these epitopes mapped to regions predicted to be hydrophilic based on Kyte and Doolittle profiles. Of the capsid epitopes identified in this study, the most immunogenic ones mapped to the C-terminal α4 helix, which lies on the solvent-exposed surface of the capsid dimer. The capsid epitopes were dengue-specific in that they could recognize antibodies in dengue virus-, but not yellow fever virus (YFV)- or Japanese encephalitis virus (JEV)-immune sera. This study has demonstrated the presence of anti-NS4a antibodies in dengue-patient sera definitively, for the first time, using authentic NS4a-derived pin-bound peptides as capture antigens. All the NS4a epitopes mapped to the amino-terminal third of the NS4a molecule. Our study suggests that the immunodominant epitopes of these two dengue proteins might have the potential to be used as a part of a recombinant multi-epitope protein containing carefully chosen E and NS1 epitopes for the detection of dengue infections with a high degree of sensitivity and specificity.

Peptides Inducing Short-Lived Antibody Responses against Plasmodium falciparum Malaria Have Shorter Structures and Are Read in a Different MHC II Functional Register

The search for a rational method of developing an antimalarial vaccine (malaria caused by Plasmodium falciparum) consists of blocking receptor-ligand interaction. Conserved peptides derived from proteins involved in invasion and having strong red blood cell binding ability have thus been identified; immunization studies using Aotus monkeys revealed that these peptides were neither immunogenic nor protection-inducing. Some of these peptides induced long-lasting and very high antibody titers and protection when their critical red blood cell binding residues were replaced to change their immunological properties. Others induced short-lived antibodies that were not associated with inducing protection. The three-dimensional structure of the short-lived antibody-inducing peptide was determined by (1)H NMR. Their HLA-DRbeta1* molecule binding ability was also determined to ascertain the relationship among three-dimensional structure, their ability to bind to major histocompatibility complex class II molecules (MHC II), and possible short-lived antibody production. These short-lived antibody-inducing peptides were 6.8 +/- 0.5 A shorter between those residues theoretically coming into contact with pocket 1 and pocket 9 of HLA-DRbeta1* molecules to which they bind than immunogenic and protection-inducing peptides. These more compact alpha-helical structures suggest that these short-lived antibody-inducing peptides could have a structure more similar to those of native peptides than immunogenic and protective ones. Such shortening was associated with a shift in HLA-DRbeta1* molecule binding and a consequent shift in functional register reading, mainly by alleles of the same haplotype when compared with immunogenic protection-inducing HABPs, suggesting an imperfect and different conformation of the MHC II peptide-TCR complex.

Evidence supporting the hypothesis that specifically modifying a malaria peptide to fit into HLA-DRβ1 *03 molecules induces antibody production and protection

EBA-175 protein is used as ligand in Plasmodium falciparum binding to erythrocytes. Evidence shows that conserved peptide 1815 from this protein having high red blood cell binding ability plays an important role in the invasion process. This peptide is neither immunogenic nor protective. Residues were substituted by amino acids having similar volume or mass but different polarity in 1815 analogues had to make them fit into HLA-DRβ1 *03 molecules; these were synthesised and inoculated into Aotus monkeys, generating different immunogenic and/or protective immune responses. A shortening in α-helix structure was found in the immunogenic and protective ones when their secondary structure was analyzed by NMR to correlate their structure with their immunological properties. This data, together with results from previous studies, suggests that this shortening in high-activity binding peptide (HABP) helical configuration may lead to better fitting into immune system molecules as shown by binding to purified HLA-DRβ1 * molecules rendering them immunogenic and protective and therefore, excellent candidates for consideration as components of a subunit based multi-component synthetic vaccine against malaria.

6746 SERA peptide analogues immunogenicity and protective efficacy against malaria is associated with short α helix formation:: Malaria protection associated with peptides α helix shortening

Erythrocyte high activity binding peptides (HABPs) have been identified for the Plasmodium falciparum serine repeat antigen (SERA). HABP 6746, located in this protein’s 50 kDa fragment had its critical binding residues replaced by amino acids having similar mass but different charge to change their immunologic properties. This peptide analogues were used to immunize Aotus monkeys that were challenged later on with a virulent P. falciparum strain to determine their protective efficacy. A shortening in α helix structure was found in the immunogenic and protective ones when their secondary structure was analyzed by NMR, to correlate their structure with their immunologic properties. These data, together with results from previous studies, suggest that this shortening in HABP helical configuration may lead to better fitting with immune system molecules, rendering them immunogenic and protective and therefore making them excellent candidates for consideration as components of a subunit based multicomponent synthetic vaccine against malaria.

Directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries.

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Biology of germinal centers in lymphoid tissue

Germinal centers in lymphoid tissue are the sites of generation of memory B cells undergoing isotype switching and somatic mutation in their Ig genes. Their formation cannot be induced by stimuli other than immunogenic ones. It seems likely that in the function and possibly also in the formation of germinal centers, one important factor is the localization of immune complexes with fixed complement on the surface of follicular dendritic cells. CD4+ T cells, located primarily in the "apical light zones" of the centers, are necessary for germinal center formation. However, their exact role in the process needs clarification, as both cell to cell contact and cytokine production could be involved at different stages of the germinal center generation. These T cells are usually specific for the antigen inducing the germinal center, but they may sometimes respond to other surface components on the B cell surface. In view of the possible stimulatory role of CD4+ T cells in follicular center-derived lymphomas, the functional significance of these T cells in germinal center proliferation is important to unravel. The B cells in germinal centers proliferate extremely rapidly, especially those located in the "dark zones." Many of them undergo apoptosis, particularly in the "basal light zones." The microenvironment of these centers is well suited to the task of expanding and selecting memory B cells of high affinity for the inducing antigen. The interactions of the proliferating B cells with dendritic cells and T cells, unevenly distributed in the various zones of the germinal center, are thought to determine which cells deserve rescue from apoptosis and induction to differentiation into small resting memory B cells. The memory B cells that emerge from the germinal center bear sIg, usually of "switched" isotype, and exhibit somatic mutations in the variable regions of their rearranged Ig genes.

Studies on the immunogenicity and tolerogenicity of T-independent antigens.

The T-cell independent immunological responses of naive mice to a series of size-fractionated dinitrophenylpolyacrylamide molecules were measured. These responses were correlated in vitro and in vivo with hapten number per molecule and hapten density per unit length. We found that to be immunogenic, a T-independent molecule must contain a threshold number (approximately 20) of appropriately spaced haptens or epitopes. We infer that this condition is necessary for simultaneous B-cell receptor binding and clustering. Molecules with less than this number of haptens are not immunogenic at any dose. Some nonimmunogenic molecules are capable of inhibiting the response to the immunogenic ones; this capability increases with increasing hapten density. In order for a molecule to accommodate 20 or more appropriately spaced haptens, it must be above a threshold size. Increasing hapten density in a molecule at or above threshold size and hapten number increases its immunogenicity. Increasing hapten density in a molecule below threshold size increases its tolerogenicity. Conclusions concerning immunogenicity and tolerogenicity of T-independent antigens may not be warranted unless the molecular weight and hapten or epitope number per molecule are well characterized.

Induction of immune reaction against rat embryonal carcinoma by activation of viral genome.

A cell line (F3/1) derived from a rat embryonal carcinoma, originally induced in fetectomized rats injected with MSV, was found to be essentially non-immunogenic in a syngeneic host; the cells did not produce C-type virions. The MSV genome could be rescued by superinfection of F3/1 cells with endogenous C-type mouse virus and the cells were converted in a producer line-F3/1-P. The produced virus was shown to be MSV by its ability to induce focus formation in mouse fibroblasts. In contrast to its non-producer ancestor, the F3/1-P line proved to be strongly immunogenic in the syngeneic host. These results indicate the possibility of converting certain non-immunogenic tumors into immunogenic ones by the activation of dormant viral genome using xenogeneic non-oncogenic helper virus.