Improvement of a DNA vaccine against BoHV-1 using chemical and molecular adjuvants
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1
Instituto Nacional de Tecnologia Agropecuaria, Instituto de Virologia, Argentina
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2
Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina
Bovine herpesvirus-1 (BoHV-1), an alphaherpesvirus, is an important etiological agent of bovine respiratory disease complex. This virus causes a wide variety of clinical manifestations that include infectious rhinotracheitis, pustular vulvo-vaginitis/balanoposthitis and neurological disease; and may predispose animals to secondary bacterial infections which lead to high morbidity and mortality. BoHV-1 uses a variety of mechanisms to elude the host’s immune system. By spreading intracellularly, it can exist in presence of virus specific antibodies, for this reason, cytotoxic T-lymphocytes (CTL) are critical for elimination of the virus.
Vaccination with conventional attenuated or inactivated vaccines has been the predominant control strategy against this virus. These vaccines neither efficiently prevent the transmission of the virus nor the establishment of latency, although they may protect individual animals against clinical disease. Even though, live-attenuated vaccines are thought to induce higher levels of protective immunity, they are not entirely safe, because they may cause abortion and latency.
DNA vaccines have emerged as an attractive approach for the generation of antigen-specific immunity. The DNA is taken up and expressed by cells resident in the tissue, and the protein is likely to be processed and presented by local antigen presenting cells. DNA vaccines mimic viral infection, thus inducing cytotoxic response without having the risk of reversion to wild type virus. However, the potency of naked DNA vaccines is limited by their inability to amplify and spread in vivo, for this reason the molecular o chemical adjuvant incorporation could be a good option.
Glycoprotein D (gD) from BoHV1 is responsable for viral adsorption and penetration in the host cells, has cytotoxic epitopes and induces neutralizing antibodies. Immune response against gD has been associated with protection previously. Adjuvants are substances capable of increase, prolong and/or potentiate immune responses.
In our group we have tested a DNA vaccine, carrying the gD sequence, with several chemical adjuvants in a BALB/c mouse model (Langellotti y col, 2011). Using this model, MONTANIDE 113101 (101)(from Seppic) was selected, according to ability to increase humoral response and IFNγ secretion. (Di Giacomo y col, 2015). On the other hand, several studies involving DNA vaccines and CD40 ligand (CD40L) have demonstrated the adjuvant capacity of this molecule.
In this report, in order to improve pCIneo-gDs vaccine, the action of 101 adjuvant and CD40L plasmid, together with a DNA vaccine encoding gD protein from BoHV-1 (pCIneo-gDs) was studied.
A dose-response curve was performed to determine the optimal concentration of gD and CD40L plasmid to be used in BALB/c mice. The selected amount was 15μg of pCIneo-gDs and 75 μg of pCD40L.
Groups of five mice were intradermally immunized on days 0 and 14 with: i)pCIneo-gDs; ii) pCIneo-gDs + pCD40L; iii) pCIneo-gDs + 101 adjuvant; iv) pCIneo-gDs + pCD40L+ 101 adjuvant; pCIneo were used as negative control.
At 30 dpv levels of antibodies against BoHV-1 in all groups were significantly higher than pCIneo-gD group (p <0.01), on the other hand, all the experimental groups presented high levels of gD antibodies (title 4.5 ± 0.5). At 14 dpv, when the specific gD IgG isotypes were determined, the group pCIneo-gDs + 101 adjuvant presented significantly higher levels of IgG1, IgG2a and IgG2b than pCIneo-gDs group (p <0.05, p <0.05, and p <0.001 respectively). On the other hand, animals immunized with pCIneo-gDs + pCD40L + 101 adjuvant showed significantly higher levels of specific IgG2a (p <0.01) and IgG2b (p <0.001) than pCIneo-gDs group. At 30 dpv although specific gD isotypes titles were high, no differences between groups were observed.
Cytokines secreted by the splenocytes stimulated in vitro with inactivated virus were measured by flow cytometry. Groups: pCIneo-gDs + pCD40L; pCIneo-gDs + 101 adjuvant and pCIneo-gDs + pCD40L + 101 adjuvant presented higher INFy levels than pCIneo-gDs group.
It is reported that when CD4 + T cells are in the presence of TGF-β and IL-6, the production of IL-17A is trigger. The IL17A stimulate several cells to secrete inflammatory mediators like IL6. On the other hand the IL6 is essential for de novo differentiation of Th17 cells. The IL17A acts on dendritic cells and is critical for optimal induction of cytotoxic lymphocytes response and protection against certain pathogens. When IL17A and IL6 were analized, animals vaccinated with pCIneo-gDs + pCD40L with or without 101 adjuvant, presented increased amount of these cytokines in comparison with pCIneo-gDs group.
Using a specific viral cytotoxicity test (JAM test), a significant increase in the cytotoxic response against BoHV-1 was detected in groups pCIneo-gDs + CD40L and pCIneo-gDs + CD40L + 101 adjuvant (consistent with the increase of IL17A) regarding pCIneo-gDs group.
On the other hand, a trial of endocytosis was carried. Dendritic cells derived from bone marrow from BALB/c mice were in vitro incubated with: plasmids pCIneo or pCIneo-gDs or pCIneo-gDs + 101 adjuvant and then the ability to endocyte Ovalbumin-FITC was measured. Dendritic cells stimulated with pCIneo-gDs + 101 adjuvant presented a significant increase in the endocytosis (p <0.05) with respect to DC stimulated only with pCIneo-gDs. This would indicate an enhancing effect of the immune response by this adjuvant.
In conclusion, the incorporation of pCD40L and 101 adjuvant to DNA vaccines induced an increased cellular response as reflected by cytokine secretion and more importantly, a cytotoxic response against BoHV-1, despite the fact that they did not induce an increase in the humoral immune response against the virus compared with that induced by the DNA vaccine alone.
All these results indicate that these adjuvants are good candidates for inclusion in a genetic vaccine and to be tested for bovines.
Acknowledgements
Acknowledgments: The authors wish to thank Dr. Osvaldo Zabal and Mrs. Teresa Morán for their valuable assistance with cell cultures, to Lic. Fatima Torales and Ms. Pamela Angelletti for technical assistance and to Seppic for providing the adjuvant.
References
Langellotti, Cecilia; Pappalardo, Juan; Quattrocchi, Valeria; Mongini, Claudia and Zamorano, Patricia. Induction of specific cytotoxic activity for bovine herpesvirus-1 by DNA immunization with different adjuvants.. Antiviral Res. 2011 90(3):134-42.
Di Giacommo; Quattrocchi, V. and Zamorano, P. Use of adjuvants to enhance the immune response 1 induced by a DNA vaccine against bovine herpesvirus-1.. Acepted for publication in Viral Immunology 2015
Keywords:
DNA vaccine,
Bovine herpes virus-1,
CD40L,
adjuvant,
mouse models
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Veterinary and Comparative Immunology
Citation:
Gammella
M,
Soria
I,
Bellusci
C,
Bidart
J,
Langellotti
C,
Quattrocchi
V and
ZAMORANO
P
(2015). Improvement of a DNA vaccine against BoHV-1 using chemical and molecular adjuvants.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00238
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Received:
29 May 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Dr. PATRICIA ZAMORANO, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina, zamorano.patricia@inta.gob.ar