High-affinity IgG Antibodies Research Articles

Immunogenicity and safety of a self-assembling ZIKV nanoparticle vaccine in mice

Zika virus (ZIKV) is a mosquito-borne flavivirus that highly susceptibly causes Guillain-Barré syndrome and microcephaly in newborns. Vaccination is one of the most effective measures for preventing infectious diseases. However, there is currently no approved vaccine to prevent ZIKV infection. Here, we developed nanoparticle (NP) vaccines by covalently conjugating self-assembled 24-subunit ferritin to the envelope structural protein subunit of ZIKV to achieve antigen polyaggregation. The immunogenicityof the NP vaccine was evaluated in mice. Compared to monomer vaccines, the NP vaccine achieved effective antigen presentation, promoted the differentiation of follicular T helper cells in lymph nodes, and induced significantly greater antigen-specific humoral and cellular immune responses. Moreover, the NP vaccine enhanced high-affinity antigen-specific IgG antibody levels, increased secretion of the cytokines IL-4 and IFN-γ by splenocytes, significantly activated T/B lymphocytes, and improved the generation of memory T/B cells. In addition, no significant adverse reactions occurred when NP vaccine was combined with adjuvants. Overall, ferritin-based NP vaccines are safe and effective ZIKV vaccine candidates.

A novel bacterium-like particles platform displaying antigens by new anchoring proteins induces efficacious immune responses.

Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant effect. It has been widely used in recent years in the development of vaccines. Existing anchoring proteins for BLP surfaces are few in number, so screening and characterization of new anchoring proteins are necessary. In this research, we created the OACD (C-terminal domain of Escherichia coli outer membrane protein A) to serve as an anchoring protein on the surface of BLP produced by the immunomodulatory bacteria Levilactobacillus brevis 23017. We used red fluorescent protein (RFP) to demonstrate the novel surface display system's effectiveness, stability, and ability to be adapted to a wide range of lactic acid bacteria. Furthermore, this study employed this surface display method to develop a novel vaccine (called COB17) by using the multi-epitope antigen of Clostridium perfringens as the model antigen. The vaccine can induce more than 50% protection rate against C. perfringens type A challenge in mice immunized with a single dose and has been tested through three routes. The vaccine yields protection rates of 75% for subcutaneous, 50% for intranasal, and 75% for oral immunization. Additionally, it elicits a strong mucosal immune response, markedly increasing levels of specific IgG, high-affinity IgG, specific IgA, and SIgA antibodies. Additionally, we used protein anchors (PA) and OACD simultaneous to show several antigens on the BLP surface. The discovery of novel BLP anchoring proteins may expand the possibilities for creating mucosal immunity subunit vaccines. Additionally, it may work in concert with PA to provide concepts for the creation of multivalent or multiple vaccines that may be used in clinical practice to treat complex illnesses.

Blood defense system - Proposal for a new concept of an immune system against blood borne pathogens comprising the liver, spleen and bone marrow.

Blood-borne pathogen (BBP) infections can rapidly progress to life-threatening sepsis and must therefore be promptly eliminated by the host's immune system. Intravascular macrophages of the liver sinusoid, splenic marginal zone and red pulp and perisinusoidal macrophage protrusions in the bone marrow (BM) directly phagocytose BBPs in the blood as an innate immune response. The liver, spleen and BM thereby work together as the blood defence system (BDS) in response to BBPs by exerting their different immunological roles. The liver removes the vast majority of these invading organisms via innate immunity, but their complete elimination is not possible without the actions of antibodies. Splenic marginal zone B cells promptly produce IgM and IgG antibodies against BBPs. The splenic marginal zone transports antigenic information from the innate to the adaptive immune systems. The white pulp of the spleen functions as adaptive immune tissue and produces specific and high-affinity antibodies with an immune memory against BBPs. The BM works to maintain immune memory by supporting the survival of memory B cells, memory T cells and long-lived plasma cells (LLPCs), all of which have dedicated niches. Furthermore, BM perisinusoidal naïve follicular B cells promptly produce IgM antibodies against BBPs in the BM sinusoid and the IgG memory B cells residing in the BM rapidly transform to plasma cells which produce high-affinity IgG antibodies upon reinfection. This review describes the complete immune defence characteristics of the BDS against BBPs through the collaboration of the liver, spleen and BM with combined different immunological roles.

LAMP-1 Chimeric to HIV-1 p55Gag in the Immunization of Neonate Mice Induces an Early Germinal Center Formation and AID Expression.

Neonates have a limited adaptive response of plasma cells, germinal center (GC) B cells, and T follicular helper cells (TFH). As neonatal vaccination can be an important tool for AIDS prevention, these limitations need to be overcome. Chimeric DNA vaccine encoding p55Gag HIV-1 protein conjugated with lysosomal-associated membrane protein 1 (LAMP-1) has been described as immunogenic in the neonate period. Herein, we investigated the immunologic mechanisms involved in neonatal immunization with a LAMP-1/p55Gag (LAMP/Gag) DNA vaccine in a C57BL/6 mouse background. Neonatal LAMP/Gag vaccination induced strong Gag-specific T-cell response until adulthood and elevated levels of anti-Gag IgG antibodies. We also demonstrated for the first time that the immunogenicity of the neonatal period with LAMP/Gag is due to the induction of high-affinity anti-p24 IgG antibodies and long-term plasma cells. Together with that, there is the generation of early TFH cells and the formation of GC sites with the upregulation of activation-induced cytidine deaminase (AID) enzyme mRNA and protein expression in draining lymph nodes after neonatal LAMP/Gag vaccination. These findings underscore that the LAMP-1 strategy in the chimeric vaccine could be useful to enhance antibody production even in the face of neonatal immaturity, and they contribute to the development of new vaccine approaches for other emerging pathogens at an early stage of life.

Changes in Anti-SARS-CoV-2 IgG Subclasses over Time and in Association with Disease Severity.

IgG is the most prominent marker of post-COVID-19 immunity. Not only does this subtype mark the late stages of infection, but it also stays in the body for a timespan of at least 6 months. However, different IgG subclasses have different properties, and their roles in specific anti-COVID-19 responses have yet to be determined. We assessed the concentrations of IgG1, IgG2, IgG3, and IgG4 against different SARS-CoV-2 antigens (N protein, S protein RBD) using a specifically designed method and samples from 348 COVID-19 patients. We noted a statistically significant association between severity of COVID-19 infection and IgG concentrations (both total and subclasses). When assessing anti-N protein and anti-RBD IgG subclasses, we noted the importance of IgG3 as a subclass. Since it is often associated with early antiviral response, we presumed that the IgG3 subclass is the first high-affinity IgG antibody to be produced during COVID-19 infection.

Inhibition of elastase enhances the adjuvanticity of alum and promotes anti–SARS-CoV-2 systemic and mucosal immunity

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.

A Genetic Approach to Display and Dissect the Cancer‐Associated O‐Glycoepitome

A hallmark of cancer is aberrant protein O-glycosylation with expression of truncated immature O-glycans including the classical Tn, STn, and T pancarcinoma antigens. The truncation of O-glycans not only present epitopes recognized by the natural low affinity antibodies to the TTn glycan haptens, which are the basis for the polyagglutination phenomena, but also expose composite aberrant O-glycopeptide epitopes that are not normally expressed and can elicit high affinity IgG antibodies. Several examples of monoclonal antibodies with high selectivity for truncated O-glycopeptide epitopes, which in effect selectively target the cancer form of O-glycoproteins, have been produced in the past. Studies demonstrate that such epitopes serve as highly cancer-specific targets potentially amenable for potent T-cell engaging strategies, however, the current insight into the abundance and distribution of aberrant O-glycopeptide epitopes and the optimal glycoforms for these remain limited. To systematically explore antibody epitopes available in the cancer-associated O-glycoproteome, we have developed a cell-based platform for display and production of human O-glycoproteins with homogeneous truncated glycoforms. This platform includes CHO and HEK293 cells stably engineered with homogeneous glycosylation capacities for the Tn, STn, and T glycoforms, and a panel of reporter expression constructs containing segments of human mucins and mucin-like domains from O-glycoproteins for display of these with different O-glycans. An overview of these efforts will be presented.

Positive selection of IgG+ over IgM+ B cells in the germinal center reaction

Positive selection of high-affinity B cells within germinal centers (GCs) drives affinity maturation of antibody responses. Here, we examined the mechanism underlying the parallel transition from immunoglobulin M (IgM) to IgG. Early GCs contained mostly unswitched IgM+ B cells; IgG+ B cells subsequently increased in frequency, dominating GC responses 14-21days after antigen challenge. Somatic hypermutation and generation of high-affinity clones occurred with equal efficiency among IgM+ and IgG+ GC B cells, and inactivation of Ig class-switch recombination did not prevent depletion of IgM+ GC B cells. Instead, high-affinity IgG+ GC B cells outcompeted high-affinity IgM+ GC B cells via a selective advantage associated with IgG antigen receptor structure but independent of the extended cytoplasmic tail. Thus, two parallel forms of GC B-cell-positive selection, based on antigen receptor variable and constant regions, respectively, operate in tandem to ensure high-affinity IgG antibodies predominate in mature serum antibody responses.

Divergent chemokine receptor expression and the consequence for human IgG4 Bcell responses.

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of Bcells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 Bcells, circulating IgG4 Bcells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive Bcells with an IgG4-inducing combination of TFH /TH2 cytokines. Consistent with these observations, we found a low abundance of IgG4 Bcells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20+ Bcell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 Bcells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.

A Genetic Approach to Display and Dissect the Cancer‐Associated O‐Glycoepitome

A hallmark of cancer is aberrant protein O‐glycosylation with expression of truncated immature O‐glycans including the classical Tn, STn, and T pancarcinoma antigens. The truncation of O‐glycans not only present epitopes recognized by the natural low affinity antibodies to the TTn glycan haptens, which are the basis for the polyagglutination phenomena, but also expose composite aberrant O‐glycopeptide epitopes that are not normally expressed and can elicit high affinity IgG antibodies. Several examples of monoclonal antibodies with high selectivity for truncated O‐glycopeptide epitopes, which in effect selectively target the cancer form of O‐glycoproteins, have been produced in the past. Studies demonstrate that such epitopes serve as highly cancer‐specific targets potentially amenable for potent T‐cell engaging strategies, however, the current insight into the abundance and distribution of aberrant O‐glycopeptide epitopes and the optimal glycoforms for these remain limited. To systematically explore antibody epitopes available in the cancer‐associated O‐glycoproteome, we have developed a cell‐based platform for display and production of human O‐glycoproteins with homogeneous truncated glycoforms. This platform includes CHO and HEK293 cells stably engineered with homogeneous glycosylation capacities for the Tn, STn, and T glycoforms, and a panel of reporter expression constructs containing segments of human mucins and mucin‐like domains from O‐glycoproteins for display of these with different O‐glycans. An overview of these efforts will be presented.Support or Funding InformationDanish National Research Foundation (DNRF107) and the Lundbeck Foundation

Immunogenicity of TNF-Inhibitors

Tumor necrosis factor inhibitors (TNFi) have significantly improved treatment outcome of rheumatic diseases since their incorporation into treatment protocols two decades ago. Nevertheless, a substantial fraction of patients experiences either primary or secondary failure to TNFi due to ineffectiveness of the drug or adverse reactions. Secondary failure and adverse events can be related to the development of anti-drug antibodies (ADA). The earliest studies that reported ADA toward TNFi mainly used drug-sensitive assays. Retrospectively, we recognize this has led to an underestimation of the amount of ADA produced due to drug interference. Drug-tolerant ADA assays also detect ADA in the presence of drug, which has contributed to the currently reported higher incidence of ADA development. Comprehension and awareness of the assay format used for ADA detection is thus essential to interpret ADA measurements correctly. In addition, a concurrent drug level measurement is informative as it may provide insight in the extent of underestimation of ADA levels and improves understanding the clinical consequences of ADA formation. The clinical effects are dependent on the ratio between the amount of drug that is neutralized by ADA and the amount of unbound drug. Pharmacokinetic modeling might be useful in this context. The ADA response generally gives rise to high affinity IgG antibodies, but this response will differ between patients. Some patients will not reach the phase of affinity maturation while others generate an enduring high titer high affinity IgG response. This response can be transient in some patients, indicating a mechanism of tolerance induction or B-cell anergy. In this review several different aspects of the ADA response toward TNFi will be discussed. It will highlight the ADA assays, characteristics and regulation of the ADA response, impact of immunogenicity on the pharmacokinetics of TNFi, clinical implications of ADA formation, and possible mitigation strategies.

AIM associated with the IgM pentamer: attackers on stand-by at aircraft carrier.

Circulating immunoglobulin M (IgM) exists in a pentameric form, possessing a polyreactive nature that responds not only to foreign antigens but also to autoantigens; thus, it is involved in both beneficial and detrimental immune responses, including protection from infection and the progression of autoimmunity. On the other hand, IgM also behaves as a carrier of the apoptosis inhibitor of macrophage (AIM) protein, storing a large amount of the inactivated form of AIM in the blood through this association. Under different disease conditions, AIM can dissociate from IgM locally or systemically to exert its function, inducing the removal of various biological debris such as excess fat, bacteria, cancer cells or dead cell debris. Most typically, upon induction of acute kidney injury (AKI), IgM-free AIM is filtered by the glomerulus in the kidney, which stimulates the clearance of intraluminal dead cells debris at the obstructed proximal tubules, thereby facilitating the repair of kidney injury. Interestingly, cats exhibit a deficiency in AIM release from IgM, which may increase their susceptibility to renal failure. Conversely, association with AIM inhibits IgM binding to the Fcα/μ receptor on follicular dendritic cells at the splenic germinal center, thereby protecting the IgM immune complex from Fcα/μ receptor-mediated internalization, which supports IgM-dependent antigen presentation to B cells and stimulates high-affinity IgG antibody production. The regulation of AIM-IgM binding, resulting from the discovery of reciprocal actions between AIM and IgM, could lead to the development of novel therapies against different diseases.

Abstract 4689: Anti-proliferative effect and tumor targeting properties of SurVaxM-derived monoclonal antibodies

Abstract Background: Survivin is an inhibitor of apoptosis protein (IAP) which is upregulated in many cancers. SurVaxM (active anti-survivin immunotherapy) is currently being evaluated in a multi-center phase II clinical trial. Circulating anti-survivin antibodies were recently detected in recurrent glioblastoma patients who received SurVaxM immunizations in a previous phase I study. Though primarily functioning as an intracellular molecule, survivin has also been identified in patient serum, and, recently, in tumor-derived exosomes. Methods: Patients in the completed SurVaxM phase I clinical trial produced both antibody and CD8+ T cell responses against survivin. In order to gauge whether antibodies to the vaccine provided any therapeutic benefit, or might serve as potential biomarkers for SurVaxM responsiveness, we tested these antibodies in pre-clinical tumor models. Murine hybridomas derived from the SurVaxM peptide were established and purified yielding several high-affinity IgG antibodies. These antibodies were characterized for target recognition and therapeutic potential. Results: Two of the clones with the highest affinities, 2C2 (IgG2a) and H30 (IgG1), recognized both SurVaxM peptide and the wild type survivin counterpart (ELISA assay), as well as endogenous, full-length survivin (western blotting). Survivin was detected on cells with these antibodies using both immunofluorescence and flow cytometry. Following s.c. implantation of glioblastoma (GL261) and melanoma (B16) tumors in immuno-competent C57BL/6 mice, administration of 2C2 and H30 antibodies (i.p. injection) reduced tumor growth in both models compared to an irrelevant IgG. An orthotopic intracranial glioma study showed similar anti-tumor effects when treated with SurVaxM-derived antibodies. Tumor growth was also inhibited in immuno-compromised (nude) animals treated with 2C2 or H30 antibodies, although to a lesser extent. Conclusions: Antibodies generated in response to the SurVaxM vaccination are highly cross-reactive to survivin and provide therapeutic benefit in immuno-competent mouse tumor models. These antibodies retain some efficacy in immuno-compromised models, indicative of a direct T-cell independent effect. An anti-tumor response through anti-survivin targeted antibodies is unexpected as surface-accessible survivin expression has not yet been well described in the literature. Data presented here highlight possible avenues for investigation of mechanism(s) of action. Citation Format: Michael J. Ciesielski, Sheila Figel, Laura Wiltsie, Phillip Galbo, Cheryl Frank, Robert A. Fenstermaker. Anti-proliferative effect and tumor targeting properties of SurVaxM-derived monoclonal antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4689. doi:10.1158/1538-7445.AM2017-4689

Abstract SY23-02: Host immune responses to HPV and HPV vaccines

Abstract Human papillomavirus (HPV) infection is the cause of multiple cancers in both women and men. There are three different licensed HPV prophylactic vaccines. Currently licensed L1 virus-like particles (VLP)–based HPV vaccines target two to nine different HPV types and have demonstrated excellent efficacy against the targeted HPV types and associated disease. Host immune responses to HPV vaccines and infection will be presented and discussed. Briefly, HPV vaccines induce nearly 100% seroconversion to all HPV types included in the vaccine. High titers of high-affinity anti-L1 IgG antibodies, to the HPV types included in the vaccine, are generated after vaccination, and these persist for several years after vaccination at levels considerably higher than those observed in natural infection. Although correlates of vaccine protection have not been identified, neutralizing antibodies are believed to be the main effectors of protection against HPV infection. Vaccine-induced antibodies are detectable not only in serum but also at mucosal sites of infection, such as the cervix and oral cavity. Local HPV-specific antibody levels, although lower, correlate well with circulating levels. Immunogenicity data have shown noninferiority of antibody responses after two doses of HPV vaccine, and the recommended number of vaccine doses was recently reduced from three to two. There is some evidence suggesting that even one dose of the bivalent HPV vaccine may provide similar protection to the recommended dose regimens. Antibody levels following one dose of the bivalent vaccine, although lower, have been shown to be stable over 4 years of follow-up and vaccine recipients remained HPV16/18 seropositive, suggesting that a single dose of the bivalent vaccine may provide durable protection against infection. The robust immunogenicity of the HPV vaccines contrasts with the immune responses observed after natural infection, in which seroconversion has been shown in only a percentage of individuals with incident HPV infection. Furthermore, levels of antibodies induced by vaccination are much higher than the levels observed after infection and appear to be preserved over time. A better understanding of long-term protection of the vaccine as well as a more comprehensive evaluation of correlates of immune protection against HPV infection are still needed. Citation Format: Ligia Pinto. Host immune responses to HPV and HPV vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY23-02. doi:10.1158/1538-7445.AM2017-SY23-02

Immunity in the spleen and blood of mice immunized with irradiated Toxoplasma gondii tachyzoites.

Toxoplasma gondii infection induces a strong and long-lasting immune response that is able to prevent most reinfections but allows tissue cysts. Irradiated, sterilized T. gondii tachyzoites are an interesting vaccine, and they induce immunity that is similar to infection, but without cysts. In this study, we evaluated the cellular immune response in the blood and spleen of mice immunized with this preparation by mouth (v.o.) or intraperitoneally (i.p.) and analyzed the protection after challenge with viable parasites. BALB/c mice were immunized with three i.p. or v.o. doses of irradiated T. gondii tachyzoites. Oral challenge with ten cysts of the ME-49 or VEG strain at 90days after the last dose resulted in high levels of protection with low parasite burden in the immunized animals. There were higher levels of specific IgG, IgA and IgM antibodies in the serum, and the i.p. immunized mice had higher levels of the high-affinity IgG and IgM antibodies than the orally immunized mice, which had more high-affinity IgA antibodies. B cells (CD19(+)), plasma cells (CD138(+)) and the CD4(+) and CD8(+) T cell populations were increased in both the blood and spleen. Cells from the spleen of the i.p. immunized mice also showed antigen-induced production of interleukin-10 (IL-10), interferon gamma (IFN-γ) and interleukin 4 (IL-4). The CD4(+) T cells, B cells and likely CD8(+) T cells from the spleens of the i.p. immunized mice proliferated with a specific antigen. The protection was correlated with the spleen and blood CD8(+) T cell, high-affinity IgG and IgM and antigen-induced IL-10 and IL-4 production. Immunization with irradiated T. gondii tachyzoites induces an immune response that is mediated by B cells and CD4(+) and CD8(+) T cells, with increased humoral and cellular immune responses that are necessary for host protection after infection. The vaccine is similar to natural infection, but free of tissue cysts; this immunity restrains infection at challenge and can be an attractive and efficient model for vaccine development in toxoplasmosis.

The respiratory syncytial virus fusion protein formulated with a novel combination adjuvant induces balanced immune responses in lambs with maternal antibodies

Respiratory syncytial virus (RSV) causes severe respiratory illness in infants. There are no licensed vaccines to prevent RSV infection. The neonate receives short-term protection from maternally derived antibodies, which, however, can also interfere with the active response to vaccination. A RSV vaccine consisting of a truncated version of the fusion protein formulated with polyI:C, innate defense regulator peptide and polyphosphazene (ΔF/TriAdj), was evaluated in two to three week-old lambs. When delivered intrapulmonary, ΔF/TriAdj elicited IgA production in the lung in addition to a robust systemic response similar to that induced by intramuscular immunization. To investigate potential interference by maternal antibodies, pregnant ewes were vaccinated with ΔF/TriAdj. Lambs born to RSV F-immune or non-immune ewes were then given three vaccinations with ΔF/TriAdj at 3 days, 4 weeks and 8 weeks post-birth. Lambs immunized intramuscularly with ΔF/TriAdj vaccine developed high-affinity ΔF-specific serum IgG and virus neutralizing antibodies, and displayed an increase in the frequency of IFN-γ-secreting cells by in vitro restimulated peripheral blood mononuclear cells. Maternal antibodies did not interfere with the development of an immune response to ΔF/TriAdj in the newborn lambs. These results indicate that immunization of neonates with ΔF/TriAdj is effective even in the face of maternal antibodies.

Development of Monoclonal Antibodies against CMP-N-Acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 1 (ST3Gal-I) Recombinant Protein Expressed inE. coli

Aberrant glycosylation is one of the major hallmarks of cancer with altered gene expression signatures of sialyltransferases. ST3Gal-I, a sialyltransferase, is known to play a crucial role in sialylation of T antigen in bladder cancer and it has reported elevated expression in breast carcinogenesis with increased tumor progression stages. The aim of the current study is to develop new monoclonal antibodies (mAbs) against human ST3Gal-I and evaluate their diagnostic potential. We developed a repertoire of stable hybridoma cell lines producing high-affinity IgG antibodies against recombinant human ST3Gal-I, expressed in E. coli BL21-DE3 strain. In order to demonstrate the diagnostic value of the mAbs, various clones were employed for the immunohistochemistry analysis of ST3Gal-I expression in cancerous tissues. Antibodies generated by 7E51C83A10 clone demonstrated a strong and specific fluorescence staining in breast cancer tissue sections and did not exhibit significant background in fibroadenoma sections. In conclusion, the mAbs raised against recombinant ST3Gal-I recognize cellular ST3Gal-I and represent a promising diagnostic tool for the immunodetection of ST3Gal-I expressing cells. Specific-reactivity of clone 7E51C83A10 mAbs towards ST3Gal-I was also confirmed by immunoblotting. Therefore, our observations warrant evaluation of ST3Gal-I as a potential marker for cancer diagnosis at larger scale.

Neutralization-Enhancing RF Antibodies for HIV Vaccines.

Efficient neutralization of HIV is a primary goal both for therapeutic and prophylactic HIV vaccines based on induction of neutralizing antibodies (NAbs) (1, 2). Neutralization capacity of NAbs correlates with their affinity to HIV-1 gp120 envelope glycoprotein (3). In HIV-infected individuals, early IgM antibodies have low affinity for gp120 glycoprotein (3), which is slightly compensated by their pentameric structure and avidity to multivalent structures of the virus. Corresponding IgG1 antibodies (3, 4), which appear later from IgM through the CSR and SHM processes (3, 5), have high affinity and specificity to gp120, but low avidity due to monomeric structure of IgG. The question is how to induce HIVspecific NAbs with both high affinity and avidity to gp120? Anti-HIV IgG antibodies 2F5 and 2G12, switched back to IgM isotype, showed increased avidity and neutralization efficacy (2). Dendritic antibody supramolecules (DAS) in one molecule combine high specificity of IgG with high avidity of IgM (6). It would be of interest to construct such a DAS with IgM as a core, carrying 10 anti-gp120 IgG monomers. Natural monomeric anti-gp120 IgG1 antibodies (3, 4) not only have low avidity insufficient for effective neutralization of HIV but may also contribute to FcR-mediated infection enhancement (7, 8). Does it mean that HIV may use high-affinity IgG antibodies for its own purposes? Fortunately, in nature there exist some helpful network-like regulatory mechanisms with a key role of RF antibodies (9). Rheumatoid factor (RF) is an autoantibody which specifically binds Fc region of IgG (10, 11). Apart from pathologic RF in rheumatoid arthritis (12), natural RF appears in many non-rheumatoid states (13). Possible beneficial physiological roles of RF include enhanced clearance of immune complexes (ICs) (14), amplification of IgG response to pathogens (15), and enhancement of virus neutralization (16, 17). RF can be induced in vivo in a highly specific way either by secondary immunization with protein antigens or in response to immunization with the newly formed ICs (10, 11). New antigenic determinants, which appear in the Fc region of IgG antibody upon antibody-antigen complex formation, may strengthen the specificity of RF (9). The level of RF was significantly higher in some of HIV-infected individuals compared to control groups (18–22). RFs were mainly IgA, IgM immunoglobulins with specificity against anti-HIV IgG (20). RF-mediated enhancement of antiHIV IgG neutralization activity was found in the sera from MCTD patients (17). Authors suggested that RF is promising for passive immunotherapy based on NAbs (17). Can RF play a key role in specific enhancement of IgG-mediated neutralization of HIV in vivo? Measurements of RF level both in long-term non-progressors (23) and dual-infected individuals (24) might give some clues. Repeated immunization of uninfected macaques with HIV1 gp120 glycoprotein may allow researchers not only to track the kinetics of RF induction but also to elucidate whether neutralization-enhancing RF antibodies can protect macaques against subsequent challenge with SHIV. Repeated immunization of SHIV-infected macaques with HIV1 gp120 glycoprotein might show whether neutralization-enhancing RF antibodies can prolong the asymptomatic period and delay the onset of AIDS. The level of RF is not stable and has a tendency to decline during the acute phase of HIV infection (22). Prolonged repeated immunizations with gp120 (e.g., immunizations every 3 weeks; the actual time between immunizations will be adjusted according to measurements of RF level in patients) would be the solution to keep the level of neutralization-enhancing RF antibodies constantly high. Singleadministration vaccine (SAV) technology, which is based on pulsatile release of gp120 from biodegradable polymeric microspheres, mimics repeated immunization scheme, and allows vaccination to be done in one shot (25, 26). Patient-specific therapeutic HIV vaccines (1) even in simplified version, using the only one gp120 variant formed after completion of HIV population homogenization process (27), can be performed via repeated immunizations from biodegradable polymeric microspheres under SAV (25, 26) technology platform. Prophylactic HIV vaccines can be based on the variations of sequential scheme (28) for prolonged pulsatile release of gp120 glycoproteins from biodegradable polymeric microspheres in single-shot (25, 26) way convenient for both patients and doctors. Studies (15, 17) have shown the high potential of neutralization-enhancing RF antibodies, but several principal questions arise:

A semisynthetic carbohydrate-lipid vaccine that protects against S. pneumoniae in mice.

Severe forms of pneumococcal meningitis, bacteraemia and pneumonia result in more than 1 million deaths each year despite the widespread introduction of carbohydrate-protein conjugate vaccines against Streptococcus pneumoniae. Here we describe a new and highly efficient antipneumococcal vaccine design based on synthetic conjugation of S. pneumoniae capsule polysaccharides to the potent lipid antigen α-galactosylceramide, which stimulates invariant natural killer T (iNKT) cells when presented by the nonpolymorphic antigen-presenting molecule CD1d. Mice injected with the new lipid-carbohydrate conjugate vaccine produced high-affinity IgG antibodies specific for pneumococcal polysaccharides. Vaccination stimulated germinal center formation; accumulation of iNKT cells with a T follicular helper cell phenotype; and increased frequency of carbohydrate-specific, long-lived memory B cells and plasmablasts. This new lipid-carbohydrate vaccination strategy induced potent antipolysaccharide immunity that protected against pneumococcal disease in mice and may also prove effective for the design of carbohydrate-based vaccines against other major bacterial pathogens.

Development and Characterization of New Monoclonal Antibodies against Human Recombinant CA XII

Carbonic anhydrases (CAs) are enzymes that catalyse the reversible hydration of CO2 to bicarbonate. CA XII is considered a potential biomarker of tumor cells and a promising target for specific therapies. The aim of the current study was to develop new monoclonal antibodies (MAbs) against human recombinant CA XII and evaluate their diagnostic potential. An extracellular catalytic domain of human CA XII was expressed in E. coli and used as an immunogen. Seven stable hybridoma cell lines producing high-affinity IgG antibodies against human CA XII were generated. The majority of MAbs were highly specific to CA XII and did not cross-react with human recombinant CA I, CA II, CA VII, and CA XIII. In order to demonstrate the diagnostic value of the MAbs, they were employed for the immunohistochemistry analysis of CA XII expression in tissues. Two MAbs (15A4 and 4A6) demonstrated a strong and specific immunostaining of CA XII in human tissue specimens. Flow cytometry analysis of 5 human tumor cell lines with the MAb 15A4 revealed its immunoreactivity with cellular CA XII. In conclusion, the MAbs raised against recombinant catalytic domain of CA XII recognize cellular CA XII and represent a promising diagnostic tool for the immunodetection of CA XII-expressing cells.