Induction of Immunogenic Response in BALB/c Mice by Live and Killed Form of Recombinant Lactococcus lactis Displaying EG95 of Echinococcus granulosus.

Background: Cystic echinococcosis is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods: The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results: Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. Mucosal IgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion: Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice.

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

Immunogenicity of HPV and HBV vaccines: adjuvanticity of synthetic analogs of monophosphoryl lipid A combined with aluminum hydroxide.

Monophosphoryl lipid A (MPL), a purified and detoxified product of lipopolysaccharide (LPS) of Salmonella minnesota R595, has been used as an adjuvant in different vaccines. In this study, the efficacy of human papillomaviruses (HPV) and hepatitis B virus (HBV) vaccines formulated with aluminum hydroxide combined with two different synthetic MPLs, 3D-(6-acyl)-PHAD or 3D-PHAD, or aluminum hydroxide combined with the mixtures of such MPLs, has been assessed. The immunogenicity in female BALB/c mice was verified by two intramuscular injections of differently formulated HPV and HBV vaccines and the total immunoglobulin G (IgG) antibody response was considered to compare the employed adjuvants. As verified experimentally, a mixture of 3D-(6-acyl)-PHAD and 3D-PHAD was able to induce significantly higher antibody titer than that of either 3D-(6-acyl)-PHAD or 3D-PHAD, when used individually. Interestingly, based on the responses achieved in terms of the total antibody levels, such mixture of synthetic MPLs was found to be even more effective than the bacterially derived MPL. Accordingly, the obtained results indicated that, if designed appropriately, synthetic MPL molecules could provide improved adjuvanticity with high level of consistency.

Antiretroviral therapy immunologic non-response in a Brazilian population: association study using pharmaco- and immunogenetic markers

ABSTRACT Background: Antiretroviral therapy (ART) saved millions from HIV-1 infection and AIDS, but some patients do not experience adequate CD4+ T cells gain despite achieving viral suppression. The genetic component of this condition is not yet completely elucidated. Objective: To identify predictive genetic markers of immune response to ART. Methods: Case-control study. Out of 176 HIV-infected patients recruited in the city of Recife, Northeast Brazil, 67 patients with no immunologic response were the cases and the remaining 109 patients who responded were the controls. A set of 94 selected single nucleotide polymorphisms (SNPs) involved in antiretroviral drugs pharmacodynamic pathways and immune system homeostasis were genotyped, while the remaining 48 were ancestry informative markers (AIMs) for controlling for eventual hidden population structure. Results: Male patients were overrepresented in non-responder group (p = 0.01). Non-responders also started with lower absolute CD4+ T cell counts (p < 0.001). We found five SNPs significantly associated with the outcome, being three more frequent in non-responders than responders: rs2243250 (IL4) A allele (p = 0.04), rs1128503 (ABCB1) A allele (p = 0.03) and rs707265 (CYP2B6) A allele (p = 0.02), whereas the other two were less frequent in non-responders: rs2069762 (IL2) C allele (p = 0.004) and rs4646437 (CYP3A4) A allele (p = 0.04). Conclusion: Some significant univariate associations remained independently associated at multivariate survival analysis modeling, such as pre-treatment CD4+ T cells counts, IL2 and ABCB1 genotypes, and use of protease inhibitors, yielding a predictive model for the probability for immune response. More studies are needed to unravel the genetic basis of ART immunological non-response.

Stress-induced cellular responses in immunogenic cell death: Implications for cancer immunotherapy.

Cancer is evading the host's defense mechanisms leading to avoidance of immune destruction. During tumor progression, immune-evading cancer cells arise due to selective pressure from the hypoxic and nutrient-deprived microenvironment. Thus, therapies aiming at re-establishing immune destruction of pathological cells constitute innovating anti-cancer strategies. Accumulating evidence suggests that selected conventional chemotherapeutic drugs increase the immunogenicity of stressed and dying cancer cells by triggering a form of cell death called immunogenic cell death (ICD), which is characterized by the release of danger-associated molecular patterns (DAMPs). In this review, we summarize the effects of ICD inducers on DAMP signaling leading to adjuvanticity and antigenicity. We will discuss the associated stress response pathways that cause the release of DAMPs leading to improved immune recognition and their relevance in cancer immunotherapy. Our aim is to highlight the contribution of adaptive immunity to the long-term clinical benefits of anticancer treatments and the properties of immune memory that can protect cancer patients against relapse.

Immunogenicity of Therapeutic Antibodies: Monitoring Antidrug Antibodies in a Clinical Context.

One of the factors that may impact drug levels of therapeutic antibodies in patients is immunogenicity, with potential loss of efficacy. Nowadays, many immunogenicity assays are available for testing antidrug antibodies (ADA). In this article, we discuss different types of immunogenicity assays and their clinical relevance in terms of drug tolerance, relation with pharmacokinetics (PK), neutralizing antibodies, potential adverse events associated with ADA, and prediction of ADA production. Drug-tolerant assays can provide insight into the process of immunogenicity, but for clinical management, these assays do not necessarily outperform drug-sensitive assays. The usefulness of any ADA assay for clinical decision making will be larger when drug concentrations are also measured, and this is true, in particular, for drug-tolerant assays.

Population Pharmacokinetics and Immunogenicity of Adalimumab in Adult Patients with Moderate-to-Severe Hidradenitis Suppurativa.

INTRODUCTION: Hidradenitis suppurativa (HS) is a serious, debilitating, chronic inflammatory skin disease. Adalimumab is a fully human, immunoglobulin G1 monoclonal antibody specific for tumor necrosis factor-alpha recently approved for use in patients with HS. The aim of this study is to describe the population pharmacokinetics and immunogenicity of adalimumab in adult patients with HS. METHODS: Data from one phase II and two phase III studies were included in the analysis. Serial serum adalimumab concentrations and anti-adalimumab antibody (AAA) development status were used to develop the population pharmacokinetic model. The population pharmacokinetic analysis involved evaluating the effects of potential covariates on adalimumab pharmacokinetics. RESULTS: Mean serum adalimumab concentrations after 40-mg weekly dosing reached steady state (10-12 µg/mL in the phase II study and 7 µg/mL in the phase III studies) by week 2 and were maintained through week 12. The percentage of patients testing positive for AAA was low (10% in the phase II study and 7% in the phase III studies). Adalimumab pharmacokinetics was described by a one-compartment model with first-order absorption. Significant covariates for clearance included the presence of AAA, baseline C-reactive protein, and baseline body weight. CONCLUSIONS: Adalimumab pharmacokinetics in HS patients was described using a one-compartment model with weight, baseline C-reactive protein, and AAA affecting adalimumab exposure. AAA development results in decreased adalimumab concentrations with a potential decrease in efficacy. Serum adalimumab concentrations in HS patients receiving 40-mg weekly dosing were similar to those observed in other indications under approved dosing regimens.

Network pharmacology of JAK inhibitors.

Small-molecule inhibitors of the Janus kinase family (JAKis) are clinically efficacious in multiple autoimmune diseases, albeit with increased risk of certain infections. Their precise mechanism of action is unclear, with JAKs being signaling hubs for several cytokines. We assessed the in vivo impact of pan- and isoform-specific JAKi in mice by immunologic and genomic profiling. Effects were broad across the immunogenomic network, with overlap between inhibitors. Natural killer (NK) cell and macrophage homeostasis were most immediately perturbed, with network-level analysis revealing a rewiring of coregulated modules of NK cell transcripts. The repression of IFN signature genes after repeated JAKi treatment continued even after drug clearance, with persistent changes in chromatin accessibility and phospho-STAT responsiveness to IFN. Thus, clinical use and future development of JAKi might need to balance effects on immunological networks, rather than expect that JAKis affect a particular cytokine response and be cued to long-lasting epigenomic modifications rather than by short-term pharmacokinetics.

Immunogenicity of anti-TNF biologic agents in the treatment of rheumatoid arthritis.

INTRODUCTION: The use of biologic disease-modifying anti-rheumatic drugs (DMARDs), including therapeutic antibodies, antibody fragments and protein constructs that target key mediators in the pathophysiology of rheumatoid arthritis (RA), has improved the chance of achieving low disease activity and clinical remission. However, individual patients respond differently to biologic DMARD therapy, particularly the tumor necrosis factor (TNF) inhibitors. AREAS COVERED: While the variation of clinical response may be related to pharmacogenetic and other unknown factors, immunogenicity associated with some of these agents may contribute in part to a lack of efficacy and immune-mediated side effects. Timely detection of immunogenicity may avoid continued administration of ineffective treatment, and reduce unnecessary risks and costs. Access to and appropriate implementation of clinically validated drug level assays is required. EXPERT OPINION: There are currently no evidence-based recommendations to guide biologic therapy on the basis of drug level and immunogenicity testing but as more data become available and better tests are developed, a strategy of immunopharmacologic guidance to individualize treatment of RA will emerge. The potential benefits of this approach must be balanced against the costs of monitoring, and further research is required.

Recombinant Immunotoxin with T-cell Epitope Mutations That Greatly Reduce Immunogenicity for Treatment of Mesothelin-Expressing Tumors.

SS1P is a recombinant immunotoxin (RIT) that targets mesothelin. It consists of an antimesothelin Fv fused to a portion of Pseudomonas exotoxin A. In clinical studies, it has produced dramatic responses in patients with advanced mesothelioma, when combined with immunosuppressive therapy so that several treatment cycles could be given. Otherwise its activity is limited by its immunogenicity. In this work, we describe the development and characterization of LMB-T20, a highly potent RIT targeted at mesothelin-expressing cancers with low immunogenicity due to removal of its eight T-cell epitopes. LMB-T20 was more active than SS1P when tested on four different mesothelin-expressing cell lines as well as on cells obtained from patients with mesothelioma. It also has potent antitumor activity in mice, and has reduced immunogenicity as measured by cytokine secretion assays. In conclusion, LMB-T20 is a favorable candidate for evaluation in clinical trials due to its reduced immunogenicity and excellent activity.

Safety and immunogenicity of 13-valent pneumococcal conjugate vaccine formulations with and without aluminum phosphate and comparison of the formulation of choice with 23-valent pneumococcal polysaccharide vaccine in elderly adults: a randomized open-label trial.

This randomized open-label trial was designed to provide preliminary immunogenicity and safety data to support development of the pediatric 13-valent pneumococcal conjugate vaccine (PCV13) for adults. The aims were to: identify an age-appropriate PCV13 formulation, i.e., with (n = 309) or without (n = 304) aluminum phosphate (AlPO 4); compare the selected PCV13 formulation (n = 309) with 23-valent pneumococcal polysaccharide vaccine (PPSV23; n = 301); and, together with an extension study, assess sequential use of pneumococcal vaccines at 1-year intervals in adults aged ≥65 years (n = 105) not pre-vaccinated with PPSV23. Immune responses were measured by ELISA and opsonophagocytic activity assays 1 month postvaccination. Immunoglobulin G responses elicited by PCV13 with AlPO 4 and PCV13 without AlPO 4 were similar for the majority, and noninferior for all PCV13 serotypes. PCV13 with AlPO 4 was generally more reactogenic, with reactions mainly mild or moderate. Thus, PCV13 with AlPO 4 (hereafter PCV13) became the selected formulation. Immune responses to PCV13 were noninferior for all but one serotype and for most PCV13 serotypes superior to PPSV23. Vaccine sequence assessments showed that for PCV13/PPSV23, the initial PCV13 dose generally enhanced responses to a subsequent PPSV23 dose, compared with PPSV23 alone. For PCV13/PCV13, a second dose did not enhance the first dose response when given after 1 year. For PCV13/PPSV23/PCV13, priming with PCV13 (vaccination 1) did not protect against lower responses induced by PPSV23 to subsequent PCV13 (vaccination 3). In conclusion, the pediatric PCV13 formulation with AlPO 4 is well tolerated and immunogenic in adults, is generally more immunogenic than PPSV23, and subsequent vaccination with PPSV23 is possible if required.

Comparison of the immunogenicity of Cervarix® and Gardasil® human papillomavirus vaccines for oncogenic non-vaccine serotypes HPV-31, HPV-33, and HPV-45 in HIV-infected adults.

Individuals infected with human immunodeficiency virus (HIV) have excess risk of developing human papillomavirus (HPV)-related disease. A substantial fraction of HPV-associated cancers is caused by HPV serotypes not included in the currently available vaccines. Among healthy women, both Cervarix(®) (HPV-16/18, GlaxoSmithKline Biologicals, GSK) and Gardasil(®) (HPV-6/11/16/18, Merck) have demonstrated partial cross-protection against certain oncogenic non-vaccine HPV-types. Currently, there are no available data on vaccine-induced cross-protection in men and little is known about cross-reactive immunity after HPV-vaccination of HIV-infected individuals. In an investigator-initiated trial, we randomized 91 HIV-positive men and women to receive vaccination with Cervarix(®) or Gardasil(®). The HPV-DNA status of the participants was determined with pcr before and after immunization. Cross-reactive antibody responses against HPV-31, HPV-33, and HPV-45 were evaluated for up to 12 months using a pseudovirion-based neutralization assay (PBNA). Geometric mean antibody titers (GMTs) were compared among vaccine groups and genders at 7 and 12 months.: Both vaccines induced anti-HPV-31, -33, and -45 neutralizing antibodies in participants who were seronegative and HPV-DNA negative for those types at study entry. Geometric mean antibody titers were comparable between vaccine groups. Interestingly, anti-HPV-31 and -33 antibody titers were higher among women compared with men at 7 and 12 months.: In conclusion, both licensed HPV-vaccines induced cross-neutralizing antibodies against frequent oncogenic non-vaccine serotypes HPV-31, HPV-33, and HPV-45 in HIV-infected adults, and women had greater serological responses against HPV-31 and -33 compared with men.

Recent advances in design of immunogenic and effective naked DNA vaccines against cancer.

A variety of clinical trials for vaccines against cancer have provided evidence that DNA vaccines are well tolerated and have an excellent safety profile. DNA vaccines require much improvement to make them sufficiently effective against cancer in the clinic. Nowadays, it is clear that an increased antigen expression correlates with improved immunogenicity and it is critical to vaccine performance in large animals and humans. Similarly, additional strategies are required to activate effective immunity against poorly immunogenic tumour antigens. This review discusses very recent scientific references focused on the development of sophisticated DNA vaccines against cancer. We report a selection of novel and relevant patents employed to improve their immunogenicity through several strategies such as the use of tissue-specific transcriptional elements, nuclear localisation signalling, codon-optimisation and by targeting antigenic proteins to secretory pathway. Recent patents validating portions or splice variants of tumour antigens as candidates for cancer DNA vaccines with improved specificity, such as mesothelin and hTERT, are also discussed. Lastly, we review novel patents on the use of genetic immunomodulators, such as "universal" T helper epitopes derived from tetanus toxin, E. coli heat labile enterotoxin and vegetable proteins, as well as cytokines, chemokines or costimulatory molecules such as IL-6, IL-15, IL- 21 to amplify immunity against cancer.

Immunogenicity of subcutaneously administered therapeutic proteins--a mechanistic perspective.

The administration of therapeutic proteins via the subcutaneous route (sc) is desired for compliance and convenience, but could be challenging due to perceived immunogenic potential or unwanted immune responses. There are clinical and preclinical data supporting as well as refuting the generalized notion that sc is more immunogenic. We provide a mechanistic perspective of immunogenicity of therapeutic proteins administered via the sc route and discuss strategies and opportunities for novel therapeutic approaches to mitigate immunogenicity.

Mir-142-3p target sequences reduce transgene-directed immunogenicity following intramuscular adeno-associated virus 1 vector-mediated gene delivery.

BACKGROUND: Muscle represents an important tissue target for adeno-associated virus (AAV) vector-mediated gene transfer in muscular, metabolic or blood-related genetic disorders. However, several studies have demonstrated the appearance of immune responses against the transgene product after intramuscular AAV vector delivery that resulted in a limited efficacy of the treatment. Use of microRNAs that are specifically expressed in antigen-presenting cells (APCs) is a promising approach for avoiding those immune responses. Cellular mir-142-3p, which is APC-specific, is able to repress the translation of its target cellular transcripts by binding to a specific target sequences. METHODS: In the present study, we explored the potential of mir-142-3p specific target sequences with respect to reducing or abolishing immune responses directed against ovalbumin (OVA), a highly immunogenic protein, expressed as transgene and delivered by AAV1 vector administered intramuscularly. RESULTS: The occurrence of immune responses against OVA transgene following intramuscular delivery by AAV have been described previously and resulted in the loss of OVA protein expression. In the present study, we demonstrate that OVA protein expression was maintained when mir-142-3pT sequences were incorporated into the expression cassette. The sustained expression of OVA protein over time correlated with a reduced increase in anti-OVA antibody levels. Furthermore, no cellular infiltrates were observed in the muscle tissue when AAV1 vectors containing four or eight repeats of mir-142-3p target sequences after the OVA sequence were used. CONCLUSIONS: The rising humoral and cellular immune responses against OVA protein after intramuscular delivery can be efficiently reduced by the use of mir-142-3p target sequences.

Immunogenicity, safety, and efficacy of abatacept administered subcutaneously with or without background methotrexate in patients with rheumatoid arthritis: results from a phase III, international, multicenter, parallel-arm, open-label study.

OBJECTIVE: To evaluate the impact of concomitant methotrexate (MTX) on subcutaneous (SC) abatacept immunogenicity, and to assess safety and efficacy. METHODS: This phase III, open-label study had a 4-month short-term (ST) period and an ongoing long-term extension (LTE) period. Rheumatoid arthritis patients were stratified to receive SC abatacept (125 mg/week) with (combination) or without MTX (monotherapy), with no intravenous loading dose; patients receiving monotherapy could add MTX in the LTE period. Immunogenicity (percentage of anti-abatacept antibody-positive patients) was assessed. ST and LTE period data are reported, including efficacy through LTE month 14 and safety through LTE month 20. RESULTS: Ninety-six of 100 enrolled patients completed the ST period; 3.9% (combination) and 4.1% of patients (monotherapy) developed transient immunogenicity, and no patients were antibody positive at month 4. Serious adverse events (SAEs) were reported in 3.9% (combination) and 6.1% of patients (monotherapy); 5.9% (combination) and 8.2% of patients (monotherapy) experienced SC injection reactions, and all were mild in intensity. Mean 28-joint Disease Activity Score (DAS28) changes were -1.67 (95% confidence interval [95% CI] -2.06, -1.28; combination) and -1.94 (95% CI -2.46, -1.42; monotherapy) at month 4. Ninety patients entered and were treated in the LTE period; 83.3% (75 of 90) remained ongoing at month 24. One LTE-treated patient (1.1%) developed immunogenicity, 14.4% of patients experienced SAEs, and no SC injection reactions were reported. For patients entering the LTE period, mean DAS28 changes from baseline were -1.84 (95% CI -2.23, -1.34; combination) and -2.86 (95% CI -3.46, -2.27; monotherapy) at month 18. CONCLUSION: SC abatacept did not elicit immunogenicity associated with loss of safety or efficacy, either with or without MTX.

Hairy cell leukaemia: biological and clinical overview from immunogenetic insights.

Hairy cell Leukaemia (HCL) is a rare neoplasm of peripheral B cells which represents a paradox in oncology. Despite its largely unknown origin and behaviour, HCL is one of the few example of dramatic success in the treatment of a malignancy. The recent steps forward to understanding the biology of HCL from immunogenetic and genomic studies have recently provided new insight into diagnosis and prognosis. Several data from immunoglobulin gene (IG) analysis have provided hints regarding the cell of origin and the ongoing selective interactions of the tumour BCR with environmental stimuli. It has also recently emerged that an unmutated status of the HCL IG can be associated with failure to respond to cladribine, genetic abnormalities indicative of poor outcome and aggressive disease. These observations suggest a central role of the tumour B-cell receptor in defining the outcome of HCL and that that IG gene analysis may have biological and prognostic relevance. Hopefully, IG analysis will help tailor treatment strategies for the most aggressive cases.