Antigen-dependent modulation of immune responses to antigen-Fc fusion proteins by Fc-effector functions.

Background: Fc-fusion proteins have been successfully developed for therapeutic purposes, but are also a promising platform for the fast generation and purification of immunogens capable of inducing strong humoral immune responses in preclinical immunization studies. As the Fc-portion of immunoglobulins fused to an antigen confers functional properties of the parental antibody, such as dimerization, binding to Fc-receptors and complement activation, several studies reported that Fc-fusion proteins elicit stronger antigen-specific antibody responses than the unfused antigen. However, dimerization or half-life extension of an antigen have also been described to enhance immunogenicity. Methods: To explore the role of Fc-effector functions for the immunogenicity of fusions proteins of viral glycoproteins and Fc fragments, the HIV-1 gp120 and the RBD of SARS-CoV-2 were fused to the wild type muIgG2a Fc fragment or mutants with impaired (LALA-PG) or improved (GASDIE) Fc-effector functions. Results: Immunization of BALB/c mice with DNA vaccines encoding gp120 - Fc LALA-PG induced significantly higher antigen-specific antibody responses than gp120 - Fc WT and GASDIE. In contrast, immunization with DNA vaccines encoding the RBD fused to the same Fc mutants, resulted in comparable anti-RBD antibody levels and similar neutralization activity against several SARS-CoV-2 variants. Conclusion: Depending on the antigen, Fc-effector functions either do not modulate or suppress the immunogenicity of DNA vaccines encoding Fc-antigen fusion proteins.

Human CD4-Binding Site Antibody Elicited by Polyvalent DNA Prime-Protein Boost Vaccine Neutralizes Cross-Clade Tier-2-HIV Strains.

The vaccine elicitation of HIV-neutralizing antibodies with tier-2-neutralization breadth has been a challenge. Here, we report the isolation and characteristics of a CD4-binding site specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent gp120 DNA prime-protein boost vaccine. HmAb64 derived from heavy chain variable germline gene IGHV1-18, light chain germline gene IGKV1-39, and had a 3rd heavy chain complementarity determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 21 (10%), including tier-2 neutralization resistant strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 bound to a conformation between prefusion closed and occluded open forms of envelope trimer, using both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4-binding site. A gp120 subunit-based vaccine can thus elicit an antibody capable of tier 2-HIV neutralization.

Comparison of Anti-rabies Virus Nucleoprotein IgY Prepared by DNA Immunization and Protein Immunization.

Immunization of egg-laying hens with viral antigens efficiently produces large amounts of virus-specific IgY antibodies from egg yolks. A supply of practical and economical antibodies against the rabies virus is being desired worldwide. We immunized hens with the antigen gene DNA of the rabies virus, purified specific IgY antibodies from the egg yolk, and characterized the immuno-protein chemistry for use as a diagnosis. To prepare specific IgY antibodies against rabies virus nucleoprotein (RV-N) by DNA immunization, laying hens were pre-injected with λ-carrageenan or Freund's complete adjuvant to increase local immune activity (pre-immune stimulation), and then immunized with RV-N recombinant plasmid DNA. RV-N-specific IgY antibodies were prepared from egg yolks of immunized hens. For comparison, conventional protein antigen immunization was also used to induce the production of RV-N-specific IgY antibodies. Laying hens were immunized with an RV-N protein antigen and RV-N-specific IgY was purified from egg yolks. The binding activity against RV-N antigens was examined using IgY samples prepared by DNA (with pre-immune stimulation) and protein immunization. Immunohistochemical staining showed that IgY antibodies prepared by protein immunization strongly detected viral antigens in the brain sections of dogs infected with the virus, whereas IgY antibodies prepared by DNA immunization did not. Enzyme-linked immunosorbent assay was performed using a commercially available rabies vaccine (inactivated virus) treated with 10% formalin and heating (60°C, 30 min and 90°C, 5 min). IgY prepared by DNA immunization had weaker reactivity with denatured antigens and lower antigen concentrations than IgY prepared by protein immunization. These results suggest that it is necessary to develop a DNA immunization method for inducing IgY antibodies against the rabies virus that strongly bind to native and denatured antigens to prepare specific IgYs that can be used for antigen detection in clinical tests.

Human Mesenchymal Stem Cells Modified with the NS5A Gene of Hepatitis C Virus Induce a Cellular Immune Response Exceeding the Response to DNA Immunization with This Gene.

Hepatitis C virus (HCV) is one of the basic culprits behind chronic liver disease, which may result in cirrhosis and hepatocarcinoma. In spite of the extensive research conducted, a vaccine against HCV has not been yet created. We have obtained human mesenchymal stem cells (hMSCs) and used them for expressing the HCV NS5A protein as a model vaccination platform. Sixteen hMSC lines of a different origin were transfected with the pcNS5A-GFP plasmid to obtain genetically modified MSCs (mMSCs). The highest efficiency was obtained by the transfection of dental pulp MSCs. C57BL/6 mice were immunized intravenously with mMSCs, and the immune response was compared with the response to the pcNS5A-GFP plasmid, which was injected intramuscularly. It was shown that the antigen-specific lymphocyte proliferation and the number of IFN-γ-synthesizing cells were two to three times higher after the mMSC immunization compared to the DNA immunization. In addition, mMSCs induced more CD4+ memory T cells and an increase in the CD4+/CD8+ ratio. The results suggest that the immunostimulatory effect of mMSCs is associated with the switch of MSCs to the pro-inflammatory phenotype and a decrease in the proportion of myeloid derived suppressor cells. Thus, the possibility of using human mMSCs for the creation of a vaccine against HCV has been shown for the first time.

[Adjuvant effect of dispersed fullerene C60 on the immune response to constructs harboring amino acid and nucleotide sequences of hepatitis C virus nonstructural NS5B protein].

INTRODUCTION: A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a weak immune response and require the use of adjuvants. The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid. MATERIALS AND METHODS: An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated. RESULTS: Mice immunization with rNS5B in a mixture with dnC60 at doses of 250 g/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 720 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60. CONCLUSIONS: Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.

RBD and Spike DNA-Based Immunization in Rabbits Elicited IgG Avidity Maturation and High Neutralizing Antibody Responses against SARS-CoV-2.

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

RBD and spike DNA-based immunization in rabbits elicited IgG avidity maturation and high neutralizing antibody responses against SARS-CoV-2

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

A Vaccine Targeting Ovine Herpesvirus 2 Glycoprotein B Protects against Sheep-Associated Malignant Catarrhal Fever.

Malignant catarrhal fever (MCF) is a complex and often fatal disease of ungulates. Effective vaccines are needed to avoid MCF outbreaks and mitigate losses. This study aimed to evaluate a sheep-associated MCF (SA-MCF) vaccine candidate targeting ovine herpesvirus 2 (OvHV-2) glycoprotein B (gB). Rabbits were used as a laboratory animal model to test the safety, immunogenicity, and protective efficacy of a chimeric virus consisting of a recombinant, non-pathogenic strain of alcelaphine herpesvirus-1 encoding OvHV-2 ORF8 to express gB (AlHV-1∆ORF73/OvHV-2-ORF8). Viral-vectored immunizations were performed by using the AlHV-1∆ORF73/OvHV-2-ORF8 chimera alone or as a DNA prime (OvHV-2-ORF8)-virus boost regimen. The viral vector was inoculated by intravenous or intramuscular routes and the DNA was delivered by intradermal shots using a gene gun. The vaccine candidates were deemed safe as no clinical signs were observed following any of the immunizations. Anti-OvHV-2 gB antibodies with neutralizing activity were induced by all immunogens. At three weeks post-final immunization, all animals were challenged intranasally with a lethal dose of OvHV-2. MCF protection rates ranging from 66.7% to 71.4% were observed in vaccinated rabbits, while all mock-vaccinated animals developed the disease. The significant protective efficacy obtained with the vaccine platforms tested in this study encourages further trials in relevant livestock species, such as cattle and bison.

Robust humoral immune response against rabies virus in rabbits and guinea pigs immunized with plasmid DNA vectors encoding rabies virus glycoproteins - An approach to the production of polyclonal antibody reagents.

DNA-based immunization has been previously shown to be an efficient approach to induce robust immunity against infectious diseases in animals and humans. The advantages of DNA vaccines are simplicity of their construction and production, low cost, high stability, and ability to elicit a full spectrum of immune responses to target antigens. The goals of this study were (i) to assess the antibody immune response to rabies virus glycoproteins (rGPs) in rabbits and guinea pigs after intramuscular immunization with pTargeT and pVAC2-mcs mammalian expression vectors encoding either the wild-type (WT) or codon-optimized (cOPT) rGP genes; and (ii) to prepare in-house rabbit anti-rGP polyclonal antibody reagents suitable for in Single Radial Immunodiffusion (SRID) and Indirect Fluorescent Antibody (IFA) assays. The maximum antibody responses against rabies virus in rabbits and guinea pigs were observed after immunization series with 500 µg/dose of pVAC2-mcs vector encoding either the WT or cOPT rGP genes adjuvanted with Emulsigen-D. No significant difference in the anti-rabies virus neutralizing antibody titers was observed in rabbits immunized with the WT and cOPT rGPs. The in-house rabbit anti-rGP polyclonal antibody reagents reacted comparable to the current reference reagents in SRID and IFA assays. The results of the study demonstrated that the DNA immunization of animals with the WT or cOPT rGPs is a promising approach to either induction of high anti-rabies virus neutralizing antibody titers in vivo or for production of polyclonal antibody reagents against rabies.

Preparation of Anti-Rabies Virus N Protein IgYs by DNA Immunization of Hens Using Different Types of Adjuvants.

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 µg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund's complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16-19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.

Llama DNA Immunization and Isolation of Functional Single-Domain Antibody Binders.

Genetic immunization is a simple, cost-effective, and powerful tool for inducing innate and adaptive immune responses to combat infectious diseases and difficult-to-treat illnesses. DNA immunization is increasingly used in the generation of monoclonal antibodies against targets for which pure proteins are unavailable or are difficult to express and purify (e.g., ion channels and receptors, transmembrane proteins, and emerging infectious pathogens). Genetic immunization has been successfully utilized in small inbred laboratory animals (mostly rodents); however, low immunogenicity of DNA/RNA injected into large mammals, including humans, is still a major challenge. Here, we provide a method for the genetic immunization of llamas, using a combination of biolistic transfection with a gene gun and intradermal injection with a DERMOJET® device, to elicit heavy-chain IgG responses against epidermal growth factor receptor (EGFR). We show the technique can be used to generate single-domain antibodies (VHHs) with nanomolar affinities to EGFR. We provide methods for gene gun bullet preparation, llama immunization, serology, phage-display library construction and panning, and VHH characterization.

Preventive Vaccination with Mesenchymal Stem Cells Protects Mice from Lethal Infection Caused by Herpes Simplex Virus 1.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect almost all organs and tissues, cause genital herpes-the most common sexually transmitted disease-disorders of the central nervous system (CNS), and lead to severe complications in children. Despite the available drugs, the incidence of HSV-1/2 continues to rise. None of the prophylactic vaccine candidates have shown a protective effect in trials nor approval for use in clinical practice. We have investigated the protective properties of mesenchymal stem cells (MSC) isolated from the bone marrow of mice. A comparative analysis of the protective response to the introduction of primary and modified MSCs (mMSC) was carried out using the plasmid containing gene of the HSV and an inactivated virus in a model of lethal HSV-1 infection in mice. mMSCs were obtained by transfection of the Us6 gene encoding glycoprotein D (gD) of the HSV, the plasmid contained the same gene. After twofold immunization with primary MSCs, the formation of antibodies interacting with the viral antigen (according to enzyme immunoassay data) and neutralizing the infectious activity of HSV-1 in the reaction of biological neutralization was observed in the peripheral blood of mice. In addition, the introduction of primary MSCs induced the production of interferon gamma (INF-γ) which is detected in the peripheral blood of mice. After infection with HSV-1, the immunized mice showed significantly increased titers of virus-specific antibodies, an increased level of IFNγ, and were completely protected from lethal HSV-1 infection. The protective effect of the other three immunogens was lower and did not exceed 50-65%. Considering the wide availability of MSCs, the proven safety of intravenous administration, and the results obtained in this work on the ability to induce innate, adaptive and protective immunity to HSV-1, MSCs can be considered a promising basis for the development of new cellular vaccines for the prevention of herpesvirus and other viral infections.

Mesenchymal Stem Cells Can Both Enhance and Inhibit the Cellular Response to DNA Immunization by Genes of Nonstructural Proteins of the Hepatitis C Virus.

Despite extensive research, there is still no vaccine against the hepatitis C virus (HCV). The aim of this study was to investigate whether MSCs can exhibit adjuvant properties during DNA vaccination against hepatitis C. We used the pcNS3-NS5B plasmid encoding five nonstructural HCV proteins and MSCs derived from mice bone marrow. Five groups of DBA mice were immunized with the plasmid and/or MSCs in a different order. Group 1 was injected with the plasmid twice at intervals of 3 weeks; Group 2 with the plasmid, and after 24 h with MSCs; Group 3 with MSCs followed by the plasmid the next day; Group 4 with only MSCs; and Group 5 with saline. When the MSCs were injected prior to DNA immunization, the cell immune response to HCV proteins assessed by the level of IFN-γ synthesis was markedly increased compared to DNA alone. In contrast, MSCs injected after DNA suppressed the immune response. Apparently, the high level of proinflammatory cytokines detected after DNA injection promotes the conversion of MSCs introduced later into the immunosuppressive MSC2. The low level of cytokines in mice before MSC administration promotes the high immunostimulatory activity of MSC1 in response to a DNA vaccine. Thus, when administered before DNA, MSCs are capable of exhibiting promising adjuvant properties.

Conformation-specific monoclonal antibodies recognizing the native structure of G protein-coupled receptor (GPCR).

It is quite difficult to generate monoclonal antibodies that recognize the three-dimensional structures of the antigens of interest. To address this limitation, we developed a new hybridoma technology termed "optimized stereospecific targeting (SST)". Here we aimed at generating stereospecific monoclonal antibodies against a G protein-coupled receptor (GPCR). The optimized SST technique enabled the efficient production of conformation-specific monoclonal antibodies against human corticotropin-releasing hormone receptor 1 (huCRHR1). Hybridoma cells secreting stereospecific monoclonal antibodies were selectively cloned by a limiting dilution method and the target monoclonal antibodies were purified by protein A column chromatography. They specifically cross-reacted with native huCRHR1 expressed on the surface of CHO cells, whereas they showed no affinity for MDA-MB-231 cancer cells, which abundantly express EphA2 on the cell surface. Furthermore, immunofluorescence analysis revealed that treatment of huCRHR1-expressing CHO cells with 4% paraformaldehyde led to a decrease in the affinity of purified monoclonal antibodies for intact huCRHR1 on the cell surface. In addition, purified monoclonal antibodies showed no cross-reactivity with huCRHR1 expressed on Sf9 insect cells. These results strongly suggest that monoclonal antibodies generated by the optimized SST technique feature specific binding to the intact form of the target GPCR on mammalian cells.

[Preventive Vaccination with Mesenchymal Stem Cells Protects Mice from Lethal Infection Caused by Herpes Simplex Virus 1].

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect almost all organs and tissues, cause genital herpes-the most common sexually transmitted disease-disorders of the central nervous system (CNS), and lead to severe complications in children. Despite the available drugs, the incidence of HSV-1/2 continues to rise. None of the prophylactic vaccine candidates have shown a protective effect in trials nor approval for use in clinical practice. We have investigated the protective properties of mesenchymal stem cells (MSC) isolated from the bone marrow of mice. A comparative analysis of the protective response to the introduction of primary and modified MSCs (mMSC) was carried out using the plasmid containing gene of the HSV and an inactivated virus in a model of lethal HSV-1 infection in mice. mMSCs were obtained by transfection of the Us6 gene encoding glycoprotein D (gD) of the HSV, the plasmid contained the same gene. After twofold immunization with primary MSCs, the formation of antibodies interacting with the viral antigen (according to enzyme immunoassay data) and neutralizing the infectious activity of HSV-1 in the reaction of biological neutralization was observed in the peripheral blood of mice. In addition, the introduction of primary MSCs induced the production of interferon gamma (INF-γ) which is detected in the peripheral blood of mice. After infection with HSV-1, the immunized mice showed significantly increased titers of virus-specific antibodies, an increased level of IFNγ, and were completely protected from lethal HSV-1 infection. The protective effect of the other three immunogens was lower and did not exceed 50-65%. Considering the wide availability of MSCs, the proven safety of intravenous administration, and the results obtained in this work on the ability to induce innate, adaptive and protective immunity to HSV-1, MSCs can be considered a promising basis for the development of new cellular vaccines for the prevention of herpesvirus and other viral infections.

M448R and MGF505-7R: Two African Swine Fever Virus Antigens Commonly Recognized by ASFV-Specific T-Cells and with Protective Potential.

African swine fever (ASF) is today's number one threat for the global swine industry. Neither commercial vaccine nor treatment is available against ASF and, thus far, only live attenuated viruses (LAV) have provided robust protection against lethal ASF virus (ASFV) challenge infections. Identification of ASFV proteins inducing protective immune responses is one of the major challenges to develop safer and efficient subunit vaccines. Immunopeptidomic studies recently performed in our laboratory allowed identifying ASFV antigens recognized by ASFV-specific CD8+ T-cells. Here, we used data from the SLAI-peptide repertoire presented by a single set of ASFV-infected porcine alveolar macrophages to generate a complex DNA vaccine composed by 15 plasmids encoding the individual peptide-bearing ORFs. DNA vaccine priming improved the protection afforded by a suboptimal dose of the BA71ΔCD2 LAV given as booster vaccination, against Georgia2007/1 lethal challenge. Interestingly, M448R was the only protein promiscuously recognized by the induced ASFV-specific T-cells. Furthermore, priming pigs with DNA plasmids encoding M488R and MGF505-7R, a CD8+ T-cell antigen previously described, confirmed these two proteins as T-cell antigens with protective potential. These studies might be useful to pave the road for designing safe and more efficient vaccine formulations in the future.

The C-Terminal Domain of Nefmut Is Dispensable for the CD8+ T Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nefmut fused at its C-terminus to viral and tumor antigens elicit a potent, effective, and anti-tolerogenic CD8+ T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nefmut-derivatives released by muscle cells. In the perspective of a possible translation into the clinic of the Nefmut-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nefmut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nefmut to associate with EVs. The EV-anchoring function was also preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. Most important, the Nefmut C-terminal deletion did not affect levels, quality, and diffusion at distal sites of the antigen-specific CD8+ T immunity. We concluded that the C-terminal Nefmut truncation does not influence stability, EV-anchoring, and CD8+ T cell immunogenicity of the fused antigen. Hence, the C-terminal deleted Nefmut may represent a safer alternative to the full-length isoform for vaccines in humans.

Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells.

Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in majority of tumors. Several TERT-based cancer vaccines are currently in clinical trials, but immune correlates of their antitumor activity remain largely unknown. Here, we characterized fine specificity and lytic potential of immune response against rat TERT in mice. BALB/c mice were primed with plasmids encoding expression-optimized hemagglutinin-tagged or nontagged TERT or empty vector and boosted with same DNA mixed with plasmid encoding firefly luciferase (Luc DNA). Injections were followed by electroporation. Photon emission from booster sites was assessed by in vivo bioluminescent imaging. Two weeks post boost, mice were sacrificed and assessed for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) production by T-cells upon their stimulation with TERT peptides and for anti-TERT antibodies. All TERT DNA-immunized mice developed cellular and antibody response against epitopes at the N-terminus and reverse transcriptase domain (rtTERT) of TERT. Photon emission from mice boosted with TERT/TERT-HA+Luc DNA was 100 times lower than from vector+Luc DNA-boosted controls. Bioluminescence loss correlated with percent of IFN-γ/IL-2/TNF-α producing CD8+ and CD4+ T-cells specific to rtTERT, indicating immune clearance of TERT/Luc-coexpressing cells. We made murine adenocarcinoma 4T1luc2 cells to express rtTERT by lentiviral transduction. Expression of rtTERT significantly reduced the capacity of 4T1luc2 to form tumors and metastasize in mice, while not affecting in vitro growth. Mice which rejected the tumors developed T-cell response against rtTERT and low/no response to the autoepitope of TERT. This advances rtTERT as key component of TERT-based therapeutic vaccines against cancer.

Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

N-Terminal Fatty Acids of NEFMUT Are Required for the CD8+ T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

We recently described a cytotoxic CD8+ T lymphocyte (CTL) vaccine platform based on the intramuscular (i.m.) injection of DNA eukaryotic vectors expressing antigens of interest fused at the C-terminus of HIV-1 Nefmut, i.e., a functionally defective mutant that is incorporated at quite high levels into exosomes/extracellular vesicles (EVs). This system has been proven to elicit strong CTL immunity against a plethora of both viral and tumor antigens, as well as inhibit both transplantable and orthotopic tumors in mice. However, a number of open issues remain regarding the underlying mechanism. Here we provide evidence that hindering the uploading into EVs of Nefmut-derived products by removing the Nefmut N-terminal fatty acids leads to a dramatic reduction of the downstream antigen-specific CD8+ T-cell activation after i.m. injection of DNA vectors in mice. This result formally demonstrates that the generation of engineered EVs is part of the mechanism underlying the in vivo induced CD8+ T-cell immunogenicity. Gaining new insights on the EV-based vaccine platform can be relevant in view of its possible translation into the clinic to counteract both chronic and acute infections as well as tumors.