Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes

Therapeutic DNA-vaccination against drug-resistant HIV-1 may hinder emergence and spread of drug-resistant HIV-1, allowing for longer successful antiretroviral treatment (ART) up-to relief of ART. We designed DNA-vaccines against drug-resistant HIV-1 based on consensus clade A integrase (IN) resistant to raltegravir: IN_in_r1 (L74M/E92Q/V151I/N155H/G163R) or IN_in_r2 (E138K/G140S/Q148K) carrying D64V abrogating IN activity. INs, overexpressed in mammalian cells from synthetic genes, were assessed for stability, route of proteolytic degradation, and ability to induce oxidative stress. Both were found safe in immunotoxicity tests in mice, with no inherent carcinogenicity: their expression did not enhance tumorigenic or metastatic potential of adenocarcinoma 4T1 cells. DNA-immunization of mice with INs induced potent multicytokine T-cell response mainly against aa 209–239, and moderate IgG response cross-recognizing diverse IN variants. DNA-immunization with IN_in_r1 protected 60% of mice from challenge with 4Tlluc2 cells expressing non-mutated IN, while DNA-immunization with IN_in_r2 protected only 20% of mice, although tumor cells expressed IN matching the immunogen. Tumor size inversely correlated with IN-specific IFN-γ/IL-2 T-cell response. IN-expressing tumors displayed compromised metastatic activity restricted to lungs with reduced metastases size. Protective potential of IN immunogens relied on their immunogenicity for CD8+ T-cells, dependent on proteasomal processing and low level of oxidative stress.

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A Novel Gene Delivery Vector of Agonistic Anti-Radioprotective 105 Expressed on Cell Membranes Shows Adjuvant Effect for DNA Immunization Against Influenza

Frontiers in Immunology ◽

10.3389/fimmu.2020.606518 ◽

2020 ◽

Vol 11 ◽

Author(s):

Tatsuya Yamazaki ◽

Mrityunjoy Biswas ◽

Kouyu Kosugi ◽

Maria Nagashima ◽

Masanori Inui ◽

...

Keyword(s):

Influenza Virus ◽

B Cells ◽

Intracellular Signaling ◽

Transmembrane Domain ◽

Lethal Dose ◽

Influenza Virus Infection ◽

Target Antigen ◽

Dna Immunization ◽

Adjuvant Effect ◽

Specific Antibodies

Radioprotective 105 (RP105) (also termed CD180) is an orphan and unconventional Toll-like receptor (TLR) that lacks an intracellular signaling domain. The agonistic anti-RP105 monoclonal antibody (mAb) can cross-link RP105 on B cells, resulting in the proliferation and activation of B cells. Anti-RP105 mAb also has a potent adjuvant effect, providing higher levels of antigen-specific antibodies compared to alum. However, adjuvanticity is required for the covalent link between anti-RP105 mAb and the antigen. This is a possible obstacle to immunization due to the link between anti-RP105 mAb and some antigens, especially multi-transmembrane proteins. We have previously succeeded in inducing rapid and potent recombinant mAbs in mice using antibody gene-based delivery. To simplify the covalent link between anti-RP105 mAb and antigens, we generated genetic constructs of recombinant anti-RP105 mAb (αRP105) bound to the transmembrane domain of the IgG-B cell receptor (TM) (αRP105-TM), which could enable the anti-RP105 mAb to link the antigen via the cell membrane. We confirmed the expression of αRP105-TM and the antigen hemagglutinin, which is a membrane protein of the influenza virus, on the same cell. We also found that αRP105-TM could activate splenic B cells, including both mature and immature cells, depending on the cell surface RP105 in vitro. To evaluate the adjuvanticity of αRP105-TM, we conducted DNA immunization in mice with the plasmids encoding αRP105-TM and hemagglutinin, followed by challenge with an infection of a lethal dose of an influenza virus. We then obtained partially but significantly hemagglutinin-specific antibodies and observed protective effects against a lethal dose of influenza virus infection. The current αRP105-TM might provide adjuvanticity for a vaccine via a simple preparation of the expression plasmids encoding αRP105-TM and of that encoding the target antigen.

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Vaccination with DNA Encoding the Immunodominant LACK Parasite Antigen Confers Protective Immunity to Mice Infected with Leishmania major

Journal of Experimental Medicine ◽

10.1084/jem.186.7.1137 ◽

1997 ◽

Vol 186 (7) ◽

pp. 1137-1147 ◽

Cited By ~ 255

Author(s):

Sanjay Gurunathan ◽

David L. Sacks ◽

Daniel R. Brown ◽

Steven L. Reiner ◽

Hughes Charest ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Protective Immunity ◽

Leishmania Major ◽

Protective Efficacy ◽

Dna Vaccination ◽

Attractive Alternative ◽

Dna Immunization ◽

Dna Encoding ◽

Ifn Γ

To determine whether DNA immunization could elicit protective immunity to Leishmania major in susceptible BALB/c mice, cDNA for the cloned Leishmania antigen LACK was inserted into a euykaryotic expression vector downstream to the cytomegalovirus promoter. Susceptible BALB/c mice were then vaccinated subcutaneously with LACK DNA and challenged with L. major promastigotes. We compared the protective efficacy of LACK DNA vaccination with that of recombinant LACK protein in the presence or absence of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic protection was achieved by LACK DNA vaccination. The control of disease progression and parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon-γ (IFN-γ) production. Moreover, both the enhancement of IFN-γ production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8+ T cells at the time of vaccination or infection also abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8+ T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.

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DNA immunization encoding the secreted nonstructural protein 3 (NS3) of hepatitis C virus and enhancing the Th1 type immune response*

Journal of Viral Hepatitis ◽

10.1046/j.1352-0504.2003.00464.x ◽

2004 ◽

Vol 11 (1) ◽

pp. 18-26 ◽

Cited By ~ 6

Author(s):

X. Jiao ◽

R. Y.-H. Wang ◽

Z. Feng ◽

G. Hu ◽

H. J. Alter ◽

...

Keyword(s):

Immune Response ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Dna Immunization ◽

Nonstructural Protein 3

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DNA immunization site determines the level of gene expression and the magnitude, but not the type of the induced immune response

PLoS ONE ◽

10.1371/journal.pone.0197902 ◽

2018 ◽

Vol 13 (6) ◽

pp. e0197902 ◽

Cited By ~ 7

Author(s):

Stefan Petkov ◽

Elizaveta Starodubova ◽

Anastasia Latanova ◽

Athina Kilpeläinen ◽

Oleg Latyshev ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Dna Immunization

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Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling

Biochemical Journal ◽

10.1042/bcj20180795 ◽

2019 ◽

Vol 476 (1) ◽

pp. 39-50 ◽

Cited By ~ 5

Author(s):

Martin A. Rossotti ◽

Kevin A. Henry ◽

Henk van Faassen ◽

Jamshid Tanha ◽

Deborah Callaghan ◽

...

Keyword(s):

Functional Characterization ◽

Growth Factor Receptor ◽

Single Domain ◽

Dna Immunization ◽

Egfr Signaling ◽

High Affinity ◽

Single Domain Antibodies ◽

Human Igg1 ◽

Protein Immunization

Abstract Up-regulation of epidermal growth factor receptor (EGFR) is a hallmark of many solid tumors, and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here, we report the isolation and functional characterization of novel camelid single-domain antibodies (sdAbs or VHHs) directed against human EGFR. The source of these VHHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost), which is notable in that genetic immunization of large, outbred animals is generally poorly effective. The VHHs targeted multiple sites on the receptor's surface with high affinity (KD range: 1–40 nM), including one epitope overlapping that of cetuximab, several epitopes conserved in the cynomolgus EGFR orthologue, and at least one epitope conserved in the mouse EGFR orthologue. Interestingly, despite their generation against human EGFR expressed from cDNA by llama cells in vivo (presumably in native conformation), the VHHs exhibited wide and epitope-dependent variation in their apparent affinities for native EGFR displayed on tumor cell lines. As fusions to human IgG1 Fc, one of the VHH-Fcs inhibited EGFR signaling induced by EGF binding with a potency similar to that of cetuximab (IC50: ∼30 nM). Thus, DNA immunization elicited high-affinity, functional sdAbs that were vastly superior to those previously isolated by our group through protein immunization.

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