Evaluations for In Vitro Correlates of Immunogenicity of Inactivated Influenza A H5, H7 and H9 Vaccines in Humans

Background

Serum antibody responses in humans to inactivated influenza A (H5N1), (H9N2) and A (H7) vaccines have been varied but frequently low, particularly for subunit vaccines without adjuvant despite hemagglutinin (HA) concentrations expected to induce good responses.

Design

To help understand the low responses to subunit vaccines, we evaluated influenza A (H5N1), (H9N2), (H7N7) vaccines and 2009 pandemic (H1N1) vaccines for antigen uptake, processing and presentation by dendritic cells to T cells, conformation of vaccine HA in antibody binding assays and gel analyses, HA titers with different red blood cells, and vaccine morphology in electron micrographs (EM).

Results

Antigen uptake, processing and presentation of H5, H7, H9 and H1 vaccine preparations evaluated in humans appeared normal. No differences were detected in antibody interactions with vaccine and matched virus; although H7 trimer was not detected in western blots, no abnormalities in the conformation of the HA antigens were identified. The lowest HA titers for the vaccines were <1∶4 for the H7 vaccine and 1∶661 for an H9 vaccine; these vaccines induced the fewest antibody responses. A (H1N1) vaccines were the most immunogenic in humans; intact virus and virus pieces were prominent in EM. A good immunogenic A (H9N2) vaccine contained primarily particles of viral membrane with external HA and NA. A (H5N1) vaccines intermediate in immunogenicity were mostly indistinct structural units with stellates; the least immunogenic A (H7N7) vaccine contained mostly small 5 to 20 nm structures.

Summary

Antigen uptake, processing and presentation to human T cells and conformation of the HA appeared normal for each inactivated influenza A vaccine. Low HA titer was associated with low immunogenicity and presence of particles or split virus pieces was associated with higher immunogenicity.     

Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA₃) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA₃ glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5₃) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5₃ preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man₉GlcNAc₂ moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man₅GlcNAc₂ moieties derived from HEK293S GnTI(−) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(−) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.     

Identification of alpha-enolase as a potential immunogenic molecule during allogeneic transplantation of human adipose-derived mesenchymal stromal cells

Background aimsGiven their low immunogenicity, immunoregulatory effects and multiple differentiation capacity, mesenchymal stromal cells (MSCs) have the potential to be used for “off-the-shelf” cell therapy to treat various diseases. However, the allorejection of MSCs indicates that they are not fully immune-privileged. In this study, the authors investigated the immunogenicity of human adipose-derived MSCs (Ad-MSCs) and identified potential immunogenic molecules.MethodsTo evaluate the immunogenicity of human Ad-MSCs in vivo, cells were transplanted into humanized mice (hu-mice), then T-cell infiltration and clearance of human Ad-MSCs were observed by immunofluorescence and bioluminescence imaging. One-way mixed lymphocyte reaction and flow cytometry were performed to evaluate the immunogenicity of human Ad-MSCs in vitro. High-throughput T-cell receptor (TCR) repertoire sequencing and mass spectrometry were applied to identified potential immunogenic molecules.ResultsThe authors observed that allogeneic Ad-MSCs recruited human T cells and caused faster clearance in hu-mice than non-humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The proliferation and activation of T cells were significantly enhanced during in vitro co-culture with human Ad-MSCs. In addition, the level of HLA-II expression on human Ad-MSCs was dramatically increased after co-culture with human peripheral blood mononuclear cells (PBMCs). High-throughput sequencing was applied to analyze the TCR repertoire of the Ad-MSC-recruited T cells to identify dominant TCR CDR3 sequences. Using synthesized TCR CDR3 peptides, the authors identified several potential immunogenic candidates, including alpha-enolase (ENO1). The ENO1 expression level of Ad-MSCs significantly increased after co-culture with PBMCs, whereas ENO1 inhibitor (ENOblock) treatment decreased the expression level of ENO1 and Ad-MSC-induced proliferation of T cells.ConclusionsThe authors’ findings improve the understanding of the immunogenicity of human Ad-MSCs and provide a theoretical basis for the safe clinical application of allogeneic MSC therapy.     

Low Immunogenicity of Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Derived from Less Immunogenic Somatic Cells

The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However, whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues, such as umbilical cord mesenchymal cells (UMCs), are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report, we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs), we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly, we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay, reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system, in T cell co-culture system as well. Furthermore, through whole genome expression microarray analysis, we showed that over 70 immune genes, including all members of HLA-I, were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation, thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine.     

A rapid Flp-In system for expression of secreted H5N1 influenza hemagglutinin vaccine immunogen in mammalian cells

Background

Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing.

Methodology/Principal Findings

We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine.

Conclusions/Significance

Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.     

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.     

Low Molecular Weight Hyaluronan-Pulsed Human Dendritic Cells Showed Increased Migration Capacity and Induced Resistance to Tumor Chemoattraction

We have shown that ex vivo pre-conditioning of bone marrow-derived dendritic cells (DC) with low molecular weight hyaluronan (LMW HA) induces antitumor immunity against colorectal carcinoma (CRC) in mice. In the present study we investigated the effects of LMW HA priming on human-tumor-pulsed monocytes-derived dendritic cells (DC/TL) obtained from healthy donors and patients with CRC. LMW HA treatment resulted in an improved maturation state of DC/TL and an enhanced mixed leucocyte reaction activity in vivo. Importantly, pre-conditioning of DC/TL with LMW HA increased their ability to migrate and reduced their attraction to human tumor derived supernatants. These effects were associated with increased CCR7 expression levels in DC. Indeed, a significant increase in migratory response toward CCL21 was observed in LMW HA primed tumor-pulsed monocyte-derived dendritic cells (DC/TL/LMW HA) when compared to LWM HA untreated cells (DC/TL). Moreover, LMW HA priming modulated other mechanisms implicated in DC migration toward lymph nodes such as the metalloproteinase activity. Furthermore, it also resulted in a significant reduction in DC migratory capacity toward tumor supernatant and IL8 in vitro. Consistently, LMW HA dramatically enhanced in vivo DC recruitment to tumor-regional lymph nodes and reduced DC migration toward tumor tissue. This study shows that LMW HA –a poorly immunogenic molecule- represents a promising candidate to improve human DC maturation protocols in the context of DC-based vaccines development, due to its ability to enhance their immunogenic properties as well as their migratory capacity toward lymph nodes instead of tumors.     

Immunogenicity and efficacy of flagellin-fused vaccine candidates targeting 2009 pandemic H1N1 influenza in mice

We have previously demonstrated that the globular head of the hemagglutinin (HA) antigen fused to flagellin of Salmonella typhimurium fljB (STF2, a TLR5 ligand) elicits protective immunity to H1N1 and H5N1 lethal influenza infections in mice (Song et al., 2008, PLoS ONE 3, e2257; Song et al., 2009, Vaccine 27, 5875–5888). These fusion proteins can be efficiently and economically manufactured in E. coli fermentation systems as next generation pandemic and seasonal influenza vaccines. Here we report immunogenicity and efficacy results of three vaccine candidates in which the HA globular head of A/California/07/2009 (H1N1) was fused to STF2 at the C-terminus (STF2.HA1), in replace of domain 3 (STF2R3.HA1), or in both positions (STF2R3.2xHA1). For all three vaccines, two subcutaneous immunizations of BALB/c mice with doses of either 0.3 or 3 µg elicit robust neutralizing (HAI) antibodies, that lead to > = 2 Log₁₀ unit reduction in day 4 lung virus titer and full protection against a lethal A/California/04/2009 challenge. Vaccination with doses as low as 0.03 µg results in partial to full protection. Each candidate, particularly the STF2R3.HA1 and STF2R3.2xHA1 candidates, elicits robust neutralizing antibody responses that last for at least 8 months. The STF2R3.HA1 candidate, which was intermediately protective in the challenge models, is more immunogenic than the H1N1 components of two commercially available trivalent inactivated influenza vaccines (TIVs) in mice. Taken together, the results demonstrate that all three vaccine candidates are highly immunogenic and efficacious in mice, and that the STF2R3.2xHA1 format is the most effective candidate vaccine format.     

Release of L-alanine by tumor cells

Culture supernatants from the weakly immunogenic T cell lymphoma L5178Y ESb were found to contain substantial amounts of alanine and lactate at a ratio of about 1:10. Supernatants from cells of the highly immunogenic mutant line ESb-D also contained lactate but only minute amounts of alanine. Moreover, ESb cells converted 14C-labeled glucose or pyruvate into labeled alanine and lactate at a ratio of about 1:10, whereas ESb-D cells yielded only labeled lactate and no detectable alanine. The injection of L-alanine in combination with L-lactate into mice strongly suppressed the capacity of their spleen cells to generate cytotoxic responses. The injection of L-alanine also suppressed the immunogenicity of ESb-D cells, as demonstrated by the generation of cytotoxic activity in vivo and by the in vivo immunization (priming) for secondary cytotoxic responses against ESb-D cells in vitro. Taken together, these experiments suggest the possibility i) that the ESb cells prevent the induction of cytotoxic responses by releasing immunosuppressive alanine, and ii) that the immunogenic mutant ESb-D may have gained immunogenicity by losing this immunosuppressive property.     

Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus

Although the human transmission of avian H5N1 virus remains low, the prevalence of this highly pathogenic infection in avian species underscores the need for a preventive vaccine that can be made without eggs. Here, we systematically analyze various forms of recombinant hemagglutinin (HA) protein for their potential efficacy as vaccines. Monomeric, trimeric, and oligomeric H5N1 HA proteins were expressed and purified from either insect or mammalian cells. The immunogenicity of different recombinant HA proteins was evaluated by measuring the neutralizing antibody response. Neutralizing antibodies to H5N1 HA were readily generated in mice immunized with the recombinant HA proteins, but they varied in potency depending on their multimeric nature and cell source. Among the HA proteins, a high-molecular-weight oligomer elicited the strongest antibody response, followed by the trimer; the monomer showed minimal efficacy. The coexpression of another viral surface protein, neuraminidase, did not affect the immunogenicity of the HA oligomer, as expected from the immunogenicity of trimers produced from insect cells. As anticipated, HA expressed in mammalian cells without NA retained the terminal sialic acid residues and failed to bind alpha2,3-linked sialic acid receptors. Taken together, these results suggest that recombinant HA proteins as individual or oligomeric trimers can elicit potent neutralizing antibody responses to avian H5N1 influenza viruses.     

Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid-core peptide technology

A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid-core peptide technology was employed. Lipid-core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1-->LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side-chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side-chains increased immunogenicity. An increase in immunogenicity (enzyme-linked immunosorbent assay (ELISA) titres) of up to 100-fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freund's adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.     

Comparative vaccine studies in HLA-A2.1-transgenic mice reveal a clustered organization of epitopes presented in hepatitis C virus natural infection

A polyepitopic CD8(+)-T-cell response is thought to be critical for control of hepatitis C virus (HCV) infection. Using transgenic mice, we analyzed the immunogenicity and dominance of most known HLA-A2.1 epitopes presented during infection by using vaccines that carry the potential to enter clinical trials: peptides, DNA, and recombinant adenoviruses. The vaccines capacity to induce specific cytotoxic T lymphocytes and interferon gamma-producing cells revealed that immunogenic epitopes are clustered in specific antigens. For two key antigens, flanking regions were shown to greatly enhance the scope of epitope recognition, whereas a DNA-adenovirus prime-boost vaccination strategy augmented epitope immunogenicity, even that of subdominant ones. The present study reveals a clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.     

Secukinumab,a novel anti–IL-17A antibody,shows low immunogenicity potential in human in vitro assays comparable to other marketed biotherapeutics with low clinical immunogenicity

Secukinumab is a human monoclonal antibody that selectively targets interleukin-17A and has been demonstrated to be highly efficacious in the treatment of moderate to severe plaque psoriasis, starting at early time points, with a sustained effect and a favorable safety profile. Biotherapeutics—including monoclonal antibodies (mAbs)—can be immunogenic, leading to formation of anti-drug antibodies (ADAs) that can result in unwanted effects, including hypersensitivity reactions or compromised therapeutic efficacy. To gain insight into possible explanations for the clinically observed low immunogenicity of secukinumab, we evaluated its immunogenicity potential by applying 2 different in vitro assays: T-cell activation and major histocompatibility complex–associated peptide proteomics (MAPPs). For both assays, monocyte-derived dendritic cells (DCs) from healthy donors were exposed in vitro to biotherapeutic proteins. DCs naturally process proteins and present the derived peptides in the context of human leukocyte antigen (HLA)-class II. HLA-DR–associated biotherapeutic-derived peptides, representing potential T–cell epitopes, were identified in the MAPPs assay. In the T-cell assay, autologous CD4⁺ T cells were co-cultured with secukinumab-exposed DCs and T-cell activation was measured by proliferation and interleukin-2 secretion. In the MAPPs analysis and T-cell activation assays, secukinumab consistently showed relatively low numbers of potential T-cell epitopes and low T-cell response rates, respectively, comparable to other biotherapeutics with known low clinical immunogenicity. In contrast, biotherapeutics with elevated clinical immunogenicity rates showed increased numbers of potential T-cell epitopes and increased T-cell response rates in T-cell activation assays, indicating an approximate correlation between in vitro assay results and clinical immunogenicity incidence.     

MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity

ABSTRACT

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method’s high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.     

Immunologic response to the influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. I. Studies in human vaccinees

Analysis of an earlier study of H3N2 and H7N2 inactivated influenza vaccines in schoolchildren demonstrated a greater viral neuraminidase (NA) immunogenicity of the vaccine containing the H7 hemagglutinin (HA) antigen to which they had not been primed, despite the lesser NA antigen content of that vaccine. Thus, prior experience with the influenza viral HA appeared to have a negative influence on immune response to NA, the associated external glycoprotein, presumably on the basis of intermolecular antigenic competition. In a second study, sequential immunologic response to influenza viral NA was compared in college students who were immunized with either conventional commercial vaccine or an antigenic reassortant H7N1 vaccine, and who subsequently experienced natural infection with an H1N1 influenza virus. Although both vaccines were only marginally immunogenic in inducing NA antibody response in seronegative subjects, in vaccinees initially seropositive for HA antibody significant NA antibody titer increases occurred with H7N1 vaccine. Subsequent natural infection boosted NA antibody less effectively in the population previously primed by natural infection than in initially seronegative subjects primed by H7N1 vaccination. It is suggested that primary immunization monospecific for influenza viral NA may alter the subsequent pattern of immune response to one more favorable to the induction of NA antibody when virus is encountered.     

Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1

Background

H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals.

Methodology/Principal Findings

We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH≤5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose₅₀. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice.

Conclusion/Significance

Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.     

Increased immunogenicity of L1210 leukemia following short-term exposure to 5(3,3′-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) in vivo or in vitro

Summary Short-term exposure of L1210 Ha leukemia to DTIC in vivo or in vitro resulted in the generation of leukemic cells that were moderately immunogenic for histocompatible (BALB/C × DBA/2)F₁ (CD2F₁) mice. In vivo treatment was carried out in the peritoneal cavity of CD2F₁ host for 8–36 h. In vitro experiments were performed in glass vessels, in which tumor cells were incubated with DTIC for 2 h at 37° C. The in vitro generation of immunogenic leukemia was conditioned by the presence of mouse liver microsomes capable of producing metabolic transformation of DTIC. It follows that the increase of tumor cell immunogenicity produced in vitro and possibly in vivo by DTIC is due to (a) metabolic product (s) that has (have) not yet been identified. Somatic mutation, selection, viral activation, or other mechanisms could be responsible for the DTIC effect. The present studies suggest that similar in vivo or in vitro techniques could be used to obtain human tumor cells with higher immunogenicity.Abbreviations AIC 5-aminoimidazole-4-carboxamide - BCNU 1,3-bis-chloroethyl nitrosourea - Cy of cyclophosphamide - DMITI drug-mediated increase tumor immunogenicity - DTIC 5(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide - MLP mouse liver preparation - PB phenobarbital     

Immune responses to highly conserved influenza A virus matrix 1 peptides

Influenza vaccine development is considered to be complicated and challenging. Constantly evolving influenza viruses require continuous global monitoring and reformulation of the vaccine strains. Peptides that are conserved among different strains and subtypes of influenza A virus are strongly considered to be attractive targets for development of cross protective influenza vaccines that stimulate cellular responses. In this study, three highly conserved (>90%) matrix 1 peptides that contain multiple T cell epitopes, ILGFVFTLTVPSERGLQRRRF (P_M1), LIRHENRMVLASTTAKA (P_M2) and LQAYQKRMGVQMQR (P_M3), were assessed for their immunogenic potential in vitro by subjecting peripheral blood mononuclear cells from healthy volunteers to repetitive stimulation with these chemically synthesised peptides and measuring their IFN‐γ concentrations, proliferation by ELISA, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, respectively. Seven samples were screened for immunogenicity of P_M1 and P_M2, and six for that of P_M3. All six samples had positive responses (IFN‐γ secretion) to P_M3 stimulation, as did five and three for P_M2 and P_M1 respectively. In contrast, seven (P_M1 and P_M2) and four (P_M3) samples showed proliferative response as compared with unstimulated cells. The encouraging immunogenic response generated by these highly conserved matrix 1 peptides indicates they are prospective candidates for development of broadly reactive influenza vaccines.

     

Epitopes and hemagglutination binding domain on subgenus B:2 adenovirus fibers.

The adenovirus fiber serves as a ligand between the virus and the host cell receptor and manifests hemagglutination (HA) activity and antigenic domains. We have screened both the antigenic and immunogenic epitopes on the adenovirus fibers of subgenus B:2 by using recombinant fiber proteins (rfibers) expressed in Escherichia coli, synthesized peptides (P1 to P8), and the corresponding antisera. The results indicated that P4 (amino acids [aa] 201 to 220), P5 (aa 231 to 250), and P7 (aa 275 to 295) presented both antigenic and immunogenic epitopes in adenovirus type 11 prototype (Ad11p), Ad34a, and Ad11a fibers. P6 (aa 251 to 270) presented both epitopes in Ad11a fiber but only an antigenic epitope in other fibers. The C-terminal 20 amino acids of the fiber, corresponding to P8, manifested an epitope of low-level immunogenicity. P5, localized at the N-terminal aa 231 to 250, displayed an epitope that reacted with fibers of all the members of subgenus B analyzed. The rfibers of Ad11p and Ad34a displayed HA activity with monkey erythrocytes, though those of Ad11a did not. Mutagenesis of the rfibers revealed that neither the fragment replacements, 11p20211a, llp26011a,and 11a28011p, nor the Ad11p rfiber with the substitutions of Tyr-260-->H (Tyr260H)and Arg279Q displayed HA activity. The Ad11a fiber knob was sensitive to proteolytic digestion, whereas that of Ad11p was resistant. The results demonstrated that the decisive HA binding domain was presented at aa 260 to 280 and was conformation dependent. Nearby amino acids, aa 283 and 284, may also affect the HA function.