A polyvalent influenza DNA vaccine applied by

needle-free intradermal delivery induces crossreactive humoral and cellular immune responses in
pigs
Marie Borggren., jens Nielsen, Ingrid Karlsson, Tina S. Dalgaard, Ramona trebblen, James A.
Williams, Aders Fomsgaard.

Vaccine 34;32 (2016);3634-40

Felipe E. Morales L.
Doctorado en Ciencias, mencin Microbiologa
Facultad de Ciencias Biolgicas
2016

Introduccin

Virus influenza endmico en cerdos

Problemas reproductivos, perdida de peso,

infecciones secundarias

Graves problemas bienestar animal

Perdida econmicas en la industria

Problemas de salud publica

Recombinacin entre virus influenza humana cerdo

Pandemia 2009 triple H1N1pdm09

Vacunas actuales contra el virus influenza porcina

basadas en virus inactivado

Inducen inmunidad contra las cepas de virus incluidos en las

vacunas

Proteccin limitada contra la diversa gama de otras cepas

circulantes

Vacunas ADN combinacin eficiente de mltiples antgenos

Optimizacin vacunas ADN para mejorar inmunogenicidad

Mejora codones

Sistema de entrega (administracin)

Vectores ADN
Vacuna polivalente ADN libre vectores antibiticos y agujas
para la administracin intradrmica en conejos
H.L. Robinson, L.A. Hunt, R.G. Vaccine, 11 (1993), pp. 957960
J.B. Ulmer, et al. Science, 259 (1993), pp. 17451749
M. Borggren, et al. Hum Vaccines Immunother, 11 (8) (2015), pp. 19831990

objetivo

Determinar la inmunogenicidad de vacuna de ADN que contiene

los genes de la influenza pandmica -1918 H1N1- hasta 1968
H3N2- y los virus influenza H1N1-pdm09., probando la induccin
de respuestas inmunes tanto humorales y celulares dirigidas
contra los antgenos homlogos y heterlogos para la vacuna

Materiales y mtodos

Construccin vacuna 6 vacunas influenza ADN

utilizando plsmido NTC9385R
M. Borggren, et al. Hum Vaccines Immunother, 11 (8) (2015), pp. 19831990

Animales y diseo experimental

22 cerdos de entre 22 das 5 semanas

Rebao libre de patgenos especficos (SPF) (Dinamarca)

Distribuidos aleatoriamente en grupos en granja de

Universidad de Aarhus.

Vacunacin cada 3 semanas 35 das

200 g vacuna (ADN)
sitio dorsal espalda

IDAL

800 g vacuna (ADN)

4 sitios de adm
6 cerdos
1972 g vacuna (ADN)
10 sitios de adm
22 cerdos

2 cerdos control

Monitoreo diario
Signos enfermedad,
T corporal
Hisopado nasal
35 das PBMC
sacrificados

2 cerdos Diluvac Forte

clulas monoculeares de sangre perifrica

Deteccin del virus influenza

Isopado nasal RT-PCR influenza A (cebador na

H1N1 y na H3N2 estacional humano)

ELISA

Medir respuesta IgG

HA: A/California/04/09(H1N1)pdm09, A/Aichi/2/1968(H3N2),

A/swine/Guangxi/13/2006(H1N2) or A/Brisbane/59/07(H1N1)

NA: A/Aichi/2/1968(H3N2)

NP :A/California/07/09(H1N1)pdm09;

M1: A/Brevig Mission/1/1918(H1N1)

M2e polypeptide

Anticuerpo IgG-anti-cerdo conjundago con peroxidasa

para deteccin

HA: hemaglutinina; NA: neuraminidasa; NP: nucleocapside: M1: protena matiz; M2e: Canal inico

Ensayo Inhibicin hemaglutinacin

Ensayo indirecto con anticuerpos especficos para

virus influenza

Metodologa segn protocolo

OMS e investigacin anterior
dos cepas porcinas:
A / porcina / Dinamarca (DK) / 10409/2013
(H1N1pdm)
A / porcina / DK / 10525/2008 (H1N2)

Ensayo microneutralizacin

Deteccin pequeas cantidades de antgenos neutralizantes

especficos (solubles) contra virus

Metodologa segn protocolo OMS e

investigacin anterior
Los virus analizados fueron:

A / California / 07/09 (H1N1pdm09)

A / Nueva Caledonia / 20/99 (H1N1)

A / porcina / DK / 10409/2013 (H1N1pdm)

100 TCID50 como inculo

clulas de rin canino Madin-Derby (MDCK)

Adaptado:
katz, Flu division CDC. Second International Influenza
seroepidemiology ECDC, Stockholm- 2011

Estimulacin de clulas monoculeares de sangre perifrica

(PBMC) y ensayos de respuesta inmune mediada por clulas
Prot. Recombinante viral

PBMC

estimuladas

enterotoxina B Staphylococcus
Control (+)

Teidas

Suero Libre
Control (-)

Citometra
Flujo

FlowJo

anti-CD3 PE-Cy7
anti-CD4 FITC
anti-CD8 PE
celulas muertas (kit violeta)
fijadas y teidas con anti-IFN-y

Resultados

resultados

Ningn cerdo mostr signos de enfermedad y el virus

no pudo ser identificado en secreciones nasales

Alta respuesta antignica

para NP y HA

Fig. 1. Influenza-specific antibody response following DNA vaccination. Pigs were vaccinated twice (arrows) i.d. with needle-free delivery with 200 g (n = 6), 800 g
(n = 6) or 1972 g (n = 5) DNA, or not DNA vaccinated at all (n = 5). Levels of IgG in the sera were measured by ELISA. Recombinant influenza proteins that were (a-d)
homologous to the vaccine or (e-h) heterologous to the vaccine were used as the coating antigens. All serum samples were tested using a fixed 1:100 or 1:125 serum
dilution. Error bars indicate the mean SEM, and significant differences from the no-vaccine control group are indicated by: ****: p < 0.0001; ***: p < 0.001; **: p < 0.01; *:
p < 0.05.

2.- La induccin de anticuerpos HI

Los cerdos vacunados tenan

anticuerpos de HI en suero por
reactividad cruzada:
cepas H1N1pdm09 y H1N2-
heterlogos para los genes de la
vacuna

Fig. 2. Serum HI antibody titers in vaccinated pigs. Pigs were vaccinated twice (arrows) i.d. with needle-free delivery with 200 g (n = 6), 800 g (n = 6) or
1972 g (n = 5) DNA, or not DNA vaccinated at all (n = 5). Vaccine-induced hemagglutination inhibition antibody responses in pig sera against (a) swine
H1N1pdm09 and (b) swine H1N2 isolates were measured. The data presented are from one representative experiment out of two performed. Error bars
indicate the mean SEM, and significant differences from the no-vaccine control group are indicated by: ****: p < 0.0001; *: p < 0.05.

3.- Induccin actividad neutralizante

Actividad
neutralizante contra
las cepas H1N1 del
virus, homlogos y
heterlogos

Fig. 3. Neutralizing activity in vaccinated pig sera. Pigs were vaccinated twice (day 0 and 21 pv1) i.d. with needle-free delivery with 200 g (n = 6), 800 g (n = 6) or
1972 g (n = 5) DNA, or not DNA vaccinated at all (n = 5). The pig sera were tested in a microneutralization assay on days 21, 28 and 35 pv1. Neutralizing antibody titers,
MN titers, were evaluated by the capacity of the sera to prevent the infection of MDCK cells by (a) H1N1pdm09, (b) 1999 H1N1 and (c) swine H1N1 isolates. The
MN titer was defined as the reciprocal dilution giving 50% infection inhibition and calculated with a linear interpolation method. Serum samples with a titer below the
detectable limit of the assay (lowest serum dilution tested was 1:20) were assigned a value of 10 for graphical representation and statistical analyses. Error bars indicate the
mean SEM, and significant differences from the no-vaccine control group are indicated by: ***: p < 0.001; **: p < 0.01; *: p < 0.05.

4.- Actividad neutralizante contra las cepas H1N1 del virus, tanto homlogos y
heterlogos
clulas CD4 + CD8contenidas niveles
ms bajos de
clulas
reestimuladas

Fig. 4. T cell sub-population IFN- response to in vitro re-stimulation with influenza proteins. Pigs were vaccinated twice (day 0 and 21pv1) i.d. with needle-free delivery with
200 g (n = 6), 800 g (n = 6) or 1972 g (n = 5) DNA, or not DNA vaccinated at all (n = 5). PBMC from the vaccinated pigs on day 35pv1 were cultured in vitro in the
presence of recombinant influenza NP 2009, NP 1918, M1 1918 and HA 2009. After 24 h, the cells were stained with anti-CD3, -CD4, -CD8 and -IFN- monoclonal
antibodies and analyzed by flow cytometry. Three T cell subsets were identified based on their CD4 and CD8 expression: (a) CD4-CD8+, (b) CD4+CD8+ and (c)
CD4+CD8- cells. Error bars indicate the mean SEM, and significant differences from the no-vaccine control group are indicated by: ***: p < 0.001; **: p < 0.01; *: p < 0.05.

Fig. 5. T cell sub-population proliferation response to in vitro re-stimulation with influenza proteins. Pigs were vaccinated twice (day 0 and 21pv1) i.d. with needle-free
delivery with 200 g (n = 6), 800 g (n = 6) or 1972 g (n = 5) DNA, or not DNA vaccinated at all (n = 5). PBMC from the vaccinated pigs on day 35pv1 were cultured
in vitro in the presence of recombinant influenza NP 2009, NP 1918, M1 1918 and HA 2009. After 6 days, the cells were stained with anti-CD3, -CD4, and -CD8
monoclonal antibodies and analyzed by flow cytometry. Three T cell subsets were identified based on their CD4 and CD8 expression: (a) CD4-CD8+, (b)
CD4+CD8+ and (c) CD4+CD8- cells. Error bars indicate the mean SEM, and significant differences from the no-vaccine control group are indicated by: **: p < 0.01;
*: p < 0.05.

Conclusiones

Se demostr que la vacuna ADN influenza polivalente, sin aguja,

induce una respuesta inmune contra antgenos homlogos y
heterlogos representada por una reactividad mediada por
anticuerpos y por clulas.
Las respuestas a la vacuna se correlaciona con la dosis de ADN, es
decir, las respuestas ms altas con dosis crecientes de ADN.
La vacunacin induce una respuesta de anticuerpos despus de la
primera vacunacin que fue impulsado despus de la revacunacin.

Adems, la vacunacin parece inducir anticuerpos reactivos

contra mltiples cepas H1N1 y H1N2 de origen humano y cerdos.
La utilizacin de antgenos superficie derivados de influenza
pandmica con antgenos internos conservados tiene la
capacidad de conferir una proteccin amplia contra las dos cepas
de virus homlogos y heterlogos .
Los resultados obtenidos desafan a optimizar la vacuna con
dosis menores de ADN que demuestren la eficacia de la vacuna
polivalente, preparando as el camino para una estrategia de
intervencin mejorada en la lucha contra la influenza.

A polyvalent influenza DNA vaccine applied by

needle-free intradermal delivery induces crossreactive humoral and cellular immune responses in
pigs
Marie Borggren., jens Nielsen, Ingrid Karlsson, Tina S. Dalgaard, Ramona trebblen, James A.
Williams, Aders Fomsgaard.

Vaccine 34;32 (2016);3634-40

Felipe E. Morales L.
Doctorado en Ciencias, mencin Microbiologa
Facultad de Ciencias Biolgicas
2016

ANEXOS

https://www.youtube.com/watch?v=Mr_g02mN1hw

Construction of the DNA vaccines

1.- Influenza DNA vaccine genes were designed from nucleotide sequences published in GenBank :
(1918 NP: A/Brevig Mission/1/18(H1N1) AY744935, 1918 M: A/Brevig Mission/1/18(H1N1) AY130766,
2009 HA: A/California/04/2009(H1N1)pdm09 ACP41105, 2009 NA: A/California/04/2009(H1N1)pdm09
ACP41107, 1968 HA: A/Aichi/2/1968(H3N2) AB295605, 1968 NA: A/Aichi/2/1968(H3N2) AB295606).
2.- The genes were made synthetically and designed to include the appropriate restriction enzymes
and Kozak sequence (GCCACC), -1 base upstream from the start codon
3.- Expression vectors: pSSI, NTC8385-VA116, and NTC9385R16
4.- The pSSI expression vector contains a kanamycin resistance gene, cytomegalovirus
immediate-early promoter, intron A and polyadenylation signal.
5.- Endogenous signal sequences from the influenza HA and NA genes were used for secretory
expression.
6.- The pSSI vaccine construct was produced in the E. coli strain DH5, using kanamycin as the
selection antibiotic.
7.- The NTC8385-VA1 (3025 bp) and the NTC9385R (1700 bp) vectors are minimalized antibioticfree plasmid vectors with the expression enhancers human T-lymphotropic virus type I (HTLVI) R region and adenovirus serotype-5 virus-associated (VA) RNA interference (only in
NTC8385-VA1).
M. Borggren, et al. Hum Vaccines Immunother, 11 (8) (2015), pp. 19831990